Signal boost cascade assay

ABSTRACT

The present disclosure relates to compositions of matter and assay methods used to detect one or more target nucleic acids of interest in a sample. The compositions and methods provide signal boost upon detection of target nucleic acids of interest in less than one minute and in some instances instantaneously at ambient temperatures down to 16° C. or less, without amplification of the target nucleic acids yet allowing for massive multiplexing, high accuracy and minimal non-specific signal generation.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 63/289,112, filed 13Dec. 2021; U.S. Ser. No. 63/359,183, filed 7 Jul. 2022; U.S. Ser. No.63/395,394, filed 5 Aug. 2022; and U.S. Ser. No. 63/397,785, filed 12Aug. 2022.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

Submitted herewith is an electronically filed sequence listing viaEFS-Web a Sequence Listing XML, entitled “LS004US1_seqlist_20221207”,created 7 Dec. 2022, which is 1,227,000 bytes in size. The sequencelisting is part of the specification of this specification and isincorporated by reference in its entirety.

PETITION UNDER 37 CFR 1.84(a)(2)

This patent application contains at least one drawing executed in color.The color drawings are necessary as the only practical medium by whichaspects of the claimed subject matter may be accurately conveyed. Theclaimed invention relates to variant proteins that alter the active sitethereof and the color drawings are necessary to easily discern thestructural difference between variants. As the color drawings are beingfiled electronically via EFS-Web, only one set of the drawings isrequired.

FIELD OF THE INVENTION

The present disclosure relates to compositions of matter and assaymethods used to detect one or more target nucleic acids of interest in asample. The compositions and methods provide a signal boost upondetection of target nucleic acids of interest in less than one minuteand at ambient temperatures down to 16° C. or less.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

Rapid and accurate identification of, e.g., infectious agents, microbecontamination, variant nucleic acid sequences that indicate the presentof diseases such as cancer or contamination by heterologous sources isimportant in order to select correct treatment; identify tainted food,pharmaceuticals, cosmetics and other commercial goods; and to monitorthe environment including identification of biothreats. Classic PCR andnucleic acid-guided nuclease or CRISPR (clustered regularly interspacedshort palindromic repeats) detection methods rely on pre-amplificationof target nucleic acids of interest to enhance detection sensitivity.However, amplification increases time to detection and may cause changesto the relative proportion of nucleic acids in samples that, in turn,lead to artifacts or inaccurate results. Improved technologies thatallow very rapid and accurate detection of nucleic acids are thereforeneeded for timely diagnosis and treatment of disease, to identify toxinsin consumables and the environment, as well as in other applications.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides compositions of matter and assay methodsto detect target nucleic acids of interest. The “nucleic acid-guidednuclease cascade assays” or “signal boost cascade assays” or “cascadeassays” described herein comprise two different ribonucleoproteincomplexes and either blocked nucleic acid molecules or blocked primermolecules. The blocked nucleic acid molecules or blocked primermolecules keep one of the ribonucleoprotein complexes “locked” unlessand until a target nucleic acid of interest activates the otherribonucleoprotein complex. The present nucleic acid-guided nucleasecascade assay can detect one or more target nucleic acids of interest(e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in lessthan one minute and in some embodiments virtually instantaneouslywithout the need for amplifying the target nucleic acid(s) of interest,thereby avoiding the drawbacks of multiplex DNA amplification, such asprimer-dimerization. Further, the cascade assay prevents “leakiness”that can lead to non-specific signal generation resulting in falsepositives by preventing unwinding of the blocked nucleic acid moleculesor blocked primer molecules (double-stranded molecules); thus, thecascade assay is quantitative in addition to being rapid. A particularlyadvantageous feature of the cascade assay is that, with the exception ofthe gRNA in RNP1, the cascade assay components are the same in eachassay no matter what target nucleic acid(s) of interest is beingdetected; moreover, the gRNA in the RNP1 is easily reprogrammed usingtraditional guide design methods.

The present disclosure is related first, to the instantaneous cascadeassay, and second, to three modalities for preventing any “leakiness” inthe cascade assay leading to false positives. The three modalitiesenhance the cascade assay and are in addition to using blocked nucleicacid molecules or blocked primer molecules in the cascade assay.

A first embodiment provides a method for identifying a target nucleicacid of interest in a sample in one minute or less at 16° C. or morecomprising the steps of: providing a reaction mixture comprising: firstribonucleoprotein complexes (RNP1s) each comprising a first nucleicacid-guided nuclease and a first gRNA, wherein the first gRNA comprisesa sequence complementary to the target nucleic acid of interest; andwherein binding of the RNP1 complex to the target nucleic acid ofinterest activates cis-cleavage and trans-cleavage activity of the firstnucleic acid-guided nuclease; second ribonucleoprotein complexes (RNP2s)comprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid of interest; wherein thesecond nucleic acid-guided nuclease optionally comprises a variantnuclease engineered such that single stranded DNA is cleaved faster thandouble stranded DNA is cleaved, wherein the variant nuclease comprisesat least one mutation to the domains that interact with the PAM regionor surrounding sequences on blocked nucleic acid molecules, and whereinthe variant nuclease exhibits both cis- and trans-cleavage activity; aplurality of the blocked nucleic acid molecules comprising a sequencecorresponding to the second gRNA, wherein the blocked nucleic acidmolecules comprise: a first region recognized by the RNP2 complex; oneor more second regions not complementary to the first region forming atleast one loop; one or more third regions complementary to andhybridized to the first region forming at least one clamp, wherein theplurality of blocked nucleic acid molecules and the RNP2s optionally areat a concentration ratio where the blocked nucleic acid molecules are atan equal or higher molar concentration than the RNP2s in the reactionmixture, wherein the blocked nucleic acid molecules optionally eachcomprise at least one bulky modification, and wherein the reactionmixture comprises at least one of a variant nuclease, the concentrationratio of the blocked nucleic acid molecules at a higher molarconcentration than the molar concentration of RNP2s in the reactionmixture, and/or the blocked nucleic acid molecules comprise at least onebulky modification; contacting the reaction mixture with the sampleunder conditions that allow the target nucleic acid of interest in thesample to bind to RNP1, wherein upon binding of the target nucleic acidof interest RNP1 becomes active initiating trans-cleavage of at leastone of the plurality of blocked nucleic acid molecules thereby producingat least one unblocked nucleic acid molecule, and wherein the at leastone unblocked nucleic acid molecule binds to RNP2 initiatingtrans-cleavage of at least one further blocked nucleic acid molecule;and detecting the cleavage products, thereby detecting the targetnucleic acid of interest in the sample in one minute or less.

An additional embodiment provides a method for identifying a targetnucleic acid of interest in a sample in one minute or less at 16° C. ormore comprising the steps of: providing a reaction mixture comprising:first ribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise afirst nucleic acid-guided nuclease and a first guide RNA (gRNA); whereinthe first gRNA comprises a sequence complementary to the nucleic acidtarget of interest, and wherein the first nucleic acid-guided nucleaseexhibits both cis-cleavage activity and trans-cleavage activity; secondribonucleoprotein complexes (RNP2s) comprising a second nucleicacid-guided nuclease and a second gRNA that is not complementary to thetarget nucleic acid of interest; wherein the second nucleic acid-guidednuclease optionally comprises a variant nuclease engineered such thatsingle stranded DNA is cleaved faster than double stranded DNA iscleaved, wherein the variant nuclease comprises at least one mutation tothe domains that interact with the PAM region or surrounding sequenceson a synthesized activating molecule, and wherein the variant nucleaseexhibits both cis- and trans-cleavage activity; a plurality of templatemolecules comprising sequence homology to the second gRNA; a pluralityof the blocked primer molecules comprising a sequence complementary tothe template molecules, wherein the blocked primer molecules cannot beextended by a polymerase, and wherein the blocked primer moleculescomprise: a first region recognized by the RNP2; one or more secondregions not complementary to the first region forming at least one loop;and one or more third regions complementary to and hybridized to thefirst region forming at least one clamp, wherein the plurality ofblocked primer molecules and the RNP2s optionally are at a concentrationratio where the blocked nucleic acid molecules are at a higher molarconcentration than the RNP2s in the reaction mixture, wherein theblocked primer molecules each optionally comprise at least one bulkymodification, and wherein the reaction mixture comprises at least one ofa variant nuclease, a concentration ratio where the blocked nucleic acidmolecules are at a higher molar concentration than the RNP2s in thereaction mixture, and/or the blocked nucleic acid molecules comprisingat least one bulky modification; and a polymerase and a plurality ofnucleotides; contacting the reaction mixture with the sample underconditions that allow nucleic acid targets of interest in the sample tobind to RNP1, wherein: upon binding of the nucleic acid targets ofinterest to the RNP1, the RNP1 becomes active trans-cleaving at leastone of the blocked primer molecules, thereby producing at least oneunblocked primer molecule that can be extended by the polymerase; the atleast one unblocked primer molecule binds to one of the templatemolecules and is extended by the polymerase and nucleotides to form atleast one synthesized activating molecule having a sequencecomplementary to the second gRNA; and the at least one synthesizedactivating molecule binds to the second gRNA, and RNP2 becomes activecleaving at least one further blocked primer molecule and at least onereporter moiety in a cascade; allowing the cascade to continue; anddetecting the unblocked primer molecules, thereby detecting the targetnucleic acid of interest in the sample in one minute or less.

Aspects of the embodiments of the methods for identifying a targetnucleic acid of interest in a sample in one minute or less can besubstituted for any assay for identifying target nucleic acids; forexample, for detecting human pathogens; animal pathogens; diseasebiomarkers; pathogens in laboratories, food processing facilities,hospitals, and in the environment, including bioterrorism applications(see the exemplary organisms listed in Tables 1, 2, 3, 5 and 6 and theexemplary human biomarkers listed in Table 4). Suitable samples fortesting include any environmental sample, such as air, water, soil,surface, food, clinical sites and products, industrial sites andproducts, pharmaceuticals, medical devices, nutraceuticals, cosmetics,personal care products, agricultural equipment and sites, and commercialsamples, and any biological sample obtained from an organism or a partthereof, such as a plant, animal (including humans), or microbe.

There is also provided in an embodiment a method of detecting a targetnucleic acid molecule in a sample in a cascade reaction comprising thesteps of: (a) providing a reaction mixture comprising: (i) a firstribonucleoprotein complex (RNP1) comprising a first nucleic acid-guidednuclease and a first guide RNA (gRNA) comprising a sequencecomplementary to a target nucleic acid molecule; (ii) a secondribonucleoprotein complex (RNP2) comprising a second nucleic acid-guidednuclease and a second gRNA that is not complementary to the targetnucleic acid molecule; and (iii) a plurality of blocked nucleic acidmolecules comprising a sequence complementary to the second guide RNA,(b) contacting the target nucleic acid molecule with the reactionmixture under conditions that, relative to a control reaction, reducethe probability of R-loop formation between the second gRNA and theplurality of blocked nucleic acid molecules, wherein: (i) upon bindingof the target nucleic acid molecule, the RNP1 becomes active wherein thefirst nucleic acid-guided nuclease cleaves at least one of the blockednucleic acid molecules, thereby producing at least one unblocked nucleicacid molecule; and (ii) at least one unblocked nucleic acid moleculebinds to the second gRNA, and the RNP2 becomes active wherein the secondnucleic acid-guided nuclease cleaves at least one further blockednucleic acid molecule; and (c) detecting the cleavage products of step(b), thereby detecting the target nucleic acid molecule in the sample.

There is also provided a second embodiment comprising a method ofincreasing the efficiency, reducing the background, increasing thesignal-to-noise ratio, reducing cis-cleavage of blocked nucleic acidmolecules and preventing unwinding of the second ribonucleoproteincomplex (RNP2) in a cascade reaction comprising: (a) a reaction mixturecomprising: (i) a first ribonucleoprotein complex (RNP1) comprising afirst nucleic acid-guided nuclease and a first guide RNA (gRNA)comprising a sequence complementary to a target nucleic acid molecule;(ii) the RNP2 comprising a second nucleic acid-guided nuclease and asecond gRNA that is not complementary to the target nucleic acidmolecule; and (iii) a plurality of blocked nucleic acid moleculescomprising a sequence complementary to the second guide RNA, and (b) thetarget nucleic acid molecule comprising a sequence complementary to thefirst gRNA; and the method comprising the step of initiating the cascadereaction by contacting (a) and (b) under conditions that reduce theprobability of R-loop formation between the blocked nucleic acidmolecules and the second gRNA, thereby reducing increasing theefficiency, reducing the background, increasing the signal-to-noiseratio, reducing cis-cleavage of blocked nucleic acid molecules andpreventing unwinding of the RNP2 relative to a control reaction.

There is also provided in a third embodiment a method of increasing thesignal-to-noise ratio in a cascade reaction comprising the steps of: (a)providing a reaction mixture comprising: (i) a first ribonucleoproteincomplex (RNP1) comprising a first nucleic acid-guided nuclease and afirst guide RNA (gRNA) comprising a sequence complementary to a targetnucleic acid molecule; (ii) a second ribonucleoprotein complex (RNP2)comprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid molecule; and (iii) aplurality of blocked nucleic acid molecules comprising a sequencecomplementary to the second guide RNA, (b) initiating the cascadereaction by contacting the target nucleic acid molecule with thereaction mixture under conditions that reduce the probability of R-loopformation between the second gRNA and the plurality of blocked nucleicacid molecules, thereby increasing the signal-to-noise ratio in thecascade reaction relative to a control reaction, wherein: (i) uponbinding of the target nucleic acid molecule, the RNP1 becomes activecleaving at least one of the blocked nucleic acid molecules, therebyproducing at least one unblocked nucleic acid molecule; and (ii) theleast one unblocked nucleic acid molecule binds to the second gRNA, andthe RNP2 becomes active cleaving at least one further blocked nucleicacid molecule; and (c) detecting the cleavage products of the cascadereaction in step (b); and (d) determining the signal-to-noise ratio ofthe cascade reactions in step (b).

A fourth embodiment provides a method of increasing the efficiency,reducing the background, increasing the signal-to-noise ratio, reducingcis-cleavage of blocked nucleic acid molecules and preventing unwindingof a second ribonucleoprotein complex (RNP2) in a cascade reactioncomprising the steps of: (a) providing a reaction mixture comprising: afirst ribonucleoprotein complex (RNP1) comprising a first nucleicacid-guided nuclease and a first guide RNA (gRNA) comprising a sequencecomplementary to a target nucleic acid molecule; the RNP2 comprising asecond nucleic acid-guided nuclease and a second gRNA that is notcomplementary to the target nucleic acid molecule; and a plurality ofblocked nucleic acid molecules comprising a sequence complementary tothe second guide RNA, (b) initiating the cascade reaction by contactingthe target nucleic acid molecule with the reaction mixture underconditions that reduce the probability of R-loop formation between thesecond gRNA and the plurality of blocked nucleic acid molecules, therebyincreasing the efficiency, reducing the background, increasing thesignal-to-noise ratio, reducing cis-cleavage of blocked nucleic acidmolecules and preventing unwinding of the RNP2 in the cascade reactionrelative to a control reaction.

In some aspects of these embodiments, the conditions that reduce R-loopformation comprise one or more of the steps of: 1) providing a molarconcentration of blocked nucleic acid molecules that exceeds the molarconcentration of ribonucleoprotein complexes; 2) engineering the nucleicacid-guided nuclease used in the ribonucleoprotein complex to result ina variant nucleic acid-guided nuclease such that single stranded DNA iscleaved faster than double stranded DNA is cleaved; and/or 3)engineering the blocked nucleic acid molecules to include bulkymodifications of a size of about 1 nm or less.

Another embodiment provides a method for preventing unwinding of blockednucleic acid molecules in the presence of an RNP in a cascade reactioncomprising the steps of: providing blocked nucleic acid molecules;providing ribonucleoprotein complexes comprising a nucleic acid-guidednuclease that exhibits both cis- and trans-cleavage activity uponactivation and a gRNA that recognizes an unblocked nucleic acid moleculeresulting from trans-cleavage of the blocked nucleic acid molecules; andproviding a molar concentration of the blocked nucleic acid moleculesthat exceeds the molar concentration of ribonucleoprotein complexes;engineering the nucleic acid-guided nuclease used in theribonucleoprotein complex to result in a variant nucleic acid-guidednuclease such that single stranded DNA is cleaved faster than doublestranded DNA is cleaved; and/or 3) engineering the blocked nucleic acidmolecules to include bulky modifications of a size of about 1 nm or lessthereby preventing unwinding of the blocked nucleic acid molecules inthe cascade reaction.

In some aspects of the aforementioned embodiments, the blocked nucleicacid molecules are blocked primer molecules.

In a further embodiment, there is provided a method for preventingunwinding of blocked nucleic acid molecules or blocked primer moleculesin the presence of an RNP comprising the steps of: providing blockednucleic acid molecules or blocked primer molecules; providingribonucleoprotein complexes comprising a nucleic acid-guided nucleasethat exhibits both cis- and trans-cleavage activity upon activation anda gRNA that recognizes an unblocked nucleic acid molecule or anunblocked primer molecule resulting from trans-cleavage of the blockednucleic acid molecule or blocked primer molecule; and providing a molarconcentration of blocked nucleic acid molecules that exceeds the molarconcentration of ribonucleoprotein complexes; engineering the nucleicacid-guided nuclease used in the ribonucleoprotein complex to result ina variant nucleic acid-guided nuclease such that single stranded DNA iscleaved times faster than double stranded DNA is cleaved; and/or 3)engineering the blocked nucleic acid molecules to include bulkymodifications of a size of about 1 nm or less.

Other embodiments provide a method for detecting target nucleic acidmolecules in a sample in less than one minute without amplifying thetarget nucleic acid molecules; and instantaneously detecting targetnucleic acid molecules in a sample without amplifying the target nucleicacid molecules.

In some aspects of the methods, the reaction mixture is provided at 16°C., and in some aspects, the reaction mixture is provided at 17° C., 18°C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27°C., 28° C., 29° C., or 30° C. or higher.

Other embodiments provide reaction mixtures for identifying a targetnucleic acid of interest in a sample in one minute or less comprising:first ribonucleoprotein (RNP1) complexes (RNP1s) each comprising a firstnucleic acid-guided nuclease and a first gRNA, wherein the first gRNAcomprises a sequence complementary to the target nucleic acid ofinterest; and wherein binding of the RNP1 complex to the target nucleicacid of interest activates cis-cleavage and trans-cleavage activity ofthe first nucleic acid-guided nuclease; second ribonucleoproteincomplexes (RNP2s) comprising a second nucleic acid-guided nuclease and asecond gRNA that is not complementary to the target nucleic acid ofinterest; wherein the second nucleic acid-guided nuclease optionallycomprises a variant nuclease engineered such that single stranded DNA iscleaved faster than double stranded DNA is cleaved, wherein the variantnuclease comprises at least one mutation to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules, and wherein the variant nuclease exhibits both cis- andtrans-cleavage activity; and a plurality of the blocked nucleic acidmolecules comprising a sequence corresponding to the second gRNA,wherein the blocked nucleic acid molecules comprise: a first regionrecognized by the RNP2 complex; one or more second regions notcomplementary to the first region forming at least one loop; one or morethird regions complementary to and hybridized to the first regionforming at least one clamp, and wherein the blocked nucleic acidmolecules optionally each comprise at least one bulky modification,wherein the plurality of blocked nucleic acid molecules and the RNP2soptionally are at a concentration ratio where blocked nucleic acidmolecules are at a higher molar concentration than the RNP2s in thereaction mixture, and wherein the reaction mixture comprises at leastone of a variant nuclease, a concentration ratio where blocked nucleicacid molecules are at a higher molar concentration than the RNP2s in thereaction mixture, and/or blocked nucleic acid molecules comprising atleast one bulky modification.

Also provided is a reaction mixture for identifying a target nucleicacid of interest in a sample in one minute or less comprising: firstribonucleoprotein complexes (RNP1s), wherein the RNP1s comprise a firstnucleic acid-guided nuclease and a first guide RNA (gRNA); wherein thefirst gRNA comprises a sequence complementary to the nucleic acid targetof interest, and wherein the first nucleic acid-guided nuclease exhibitsboth cis-cleavage activity and trans-cleavage activity; secondribonucleoprotein complexes (RNP2s) comprising a second nucleicacid-guided nuclease and a second gRNA that is not complementary to thetarget nucleic acid of interest; wherein the second nucleic acid-guidednuclease optionally comprises a variant nuclease engineered such thatsingle stranded DNA is cleaved faster than double stranded DNA iscleaved, wherein the variant nuclease comprises at least one mutation tothe domains that interact with the PAM region or surrounding sequenceson synthesized activating molecules, and wherein the variant nucleaseexhibits both cis- and trans-cleavage activity; a plurality of templatemolecules comprising sequence homology to the second gRNA; a pluralityof the blocked primer molecules comprising a sequence complementary tothe template molecules, wherein the blocked primer molecules cannot beextended by a polymerase, and wherein the blocked primer moleculescomprise: a first region recognized by the RNP2; one or more secondregions not complementary to the first region forming at least one loop;and one or more third regions complementary to and hybridized to thefirst region forming at least one clamp, wherein the blocked primermolecules optionally each comprise at least one bulky modification andwherein the plurality of blocked primer molecules and the RNP2soptionally are at a concentration ratio where blocked nucleic acidmolecules are at a higher molar concentration than the RNP2s in thereaction mixture, and wherein the reaction mixture comprises at leastone of a variant nuclease, at a concentration ratio where blockednucleic acid molecules are at a higher molar concentration than theRNP2s in the reaction mixture, and/or blocked nucleic acid moleculescomprising at least one bulky modification; and a polymerase and aplurality of nucleotides.

Further provided is a composition of matter comprising:ribonucleoprotein complexes (RNPs) comprising a nucleic acid-guidednuclease and a gRNA that is not complementary to the target nucleic acidof interest; wherein the nucleic acid-guided nuclease optionallycomprises a variant nuclease engineered such that single stranded DNA iscleaved faster than double stranded DNA is cleaved, wherein the variantnuclease comprises at least one mutation to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules, and wherein the variant nuclease exhibits both cis- andtrans-cleavage activity; and a plurality of the blocked nucleic acidmolecules comprising a sequence corresponding to the gRNA, wherein theblocked nucleic acid molecules comprise: a first region recognized bythe RNP complex; one or more second regions not complementary to thefirst region forming at least one loop; one or more third regionscomplementary to and hybridized to the first region forming at least oneclamp, wherein the blocked nucleic acid molecules each comprise at leastone bulky modification, wherein the blocked nucleic acid moleculesoptionally each comprise at least one bulky modification, and whereinthe plurality of blocked nucleic acid molecules and the RNP2s optionallyare at a concentration ratio where the blocked nucleic acid moleculesare at a higher molar concentration than the RNP2s in the reactionmixture, and wherein the composition comprises at least one of a variantnuclease, a concentration ratio where the blocked nucleic acid moleculesare at a higher molar concentration than the RNP2s in the reactionmixture, and/or blocked nucleic acid molecules comprising at least onebulky modification; and a polymerase and a plurality of nucleotides.

Additionally provided is a composition of matter comprising:ribonucleoprotein complexes (RNPs) comprising a nucleic acid-guidednuclease and a gRNA that is not complementary to the target nucleic acidof interest; wherein the second nucleic acid-guided nuclease optionallycomprises a variant nuclease engineered such that single stranded DNA iscleaved faster than double stranded DNA is cleaved, wherein the variantnuclease comprises at least one mutation to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules, and wherein the variant nuclease exhibits both cis- andtrans-cleavage activity; a plurality of template molecules comprisingsequence homology to the gRNA; and a plurality of the blocked primermolecules comprising a sequence complementary to the template molecules,wherein the blocked primer molecules cannot be extended by a polymerase,and wherein the blocked primer molecules comprise: a first regionrecognized by the RNP2; one or more second regions not complementary tothe first region forming at least one loop; and one or more thirdregions complementary to and hybridized to the first region forming atleast one clamp, wherein the blocked primer molecules optionally eachcomprise at least one bulky modification, and wherein the plurality ofblocked primer molecules and the RNPs optionally are at a concentrationwhere the blocked nucleic acid molecules are at a molar concentrationequal to or greater than the molar concentration of the RNPs in thereaction mixture, and wherein the composition comprises at least one ofa variant nuclease, a concentration ratio where blocked nucleic acidmolecules are at a higher molar concentration than the RNP2s in thereaction mixture, and/or blocked nucleic acid molecules comprising atleast one bulky modification; and a polymerase and a plurality ofnucleotides.

In some aspects of these embodiments, the reaction mixture furthercomprises reporter moieties, wherein the reporter moieties produce adetectable signal upon trans-cleavage activity by the RNP2 to identifythe presence of one or more nucleic acid targets of interest in thesample. In some aspects, the reporter moieties are not coupled to theblocked primer molecules, and wherein upon cleavage by RNP2, a signalfrom the reporter moiety is detected; yet in other aspects, the reportermoieties are coupled to the blocked primer molecules, and wherein uponcleavage by RNP2, a signal from the reporter moiety is detected.

In some aspects of all embodiments comprising bulky modifications, thebulky modifications are about 1 nm in size, and in some aspects, thebulky modifications are about 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm,0.4 nm, 0.3 nm, 0.2 nm, or 0.1 nm in size. In some aspects, the bulkymodifications are about 0.9 nm, 0.8 nm, 0.7 nm, 0.6 nm, 0.5 nm, 0.4 nm,0.3 nm, 0.2 nm, or 0.1 nm in size. In some aspects, the blocked nucleicacid molecules include bulky modifications and wherein there are twobulky modifications with one bulky modification located on the 5′ end ofthe blocked nucleic acid molecule and one bulky modification located onthe 3′ end of the blocked nucleic acid molecule, and where the 5′ and 3′ends comprising the two bulky modifications are less than 11 nm from oneanother. In other aspects, the bulky modification is on a 5′ end ofblocked nucleic acid molecules and may be selected from the group of 5′Fam (6-fluorescein amidite); Black Hole Quencher-1-5′; biotin TEG (15atom triethylene glycol spacer); biotin-5′; and cholesterol TEG (15 atomtriethylene glycol spacer). In other aspects, the bulky modification ison a 3′ end of the blocked nucleic acid molecules and may be selectedfrom the group of Black Hole Quencher-1-3′; biotin-3′; and TAMRA-3′(carboxytetramethylrhodamine). In some aspects, a bulky modification isbetween two internal nucleic acid residues of the blocked nucleic acidmolecules and may be selected from the group of Cy3 internal and Cy5,and in some aspects, the bulky modification is an internal nucleotidebase modification and may be selected from the group of biotindeoxythymidine dT; disthiobiotin NHS; and fluorescein dT.

In some aspects of these embodiments, the blocked nucleic acid moleculesor blocked primer molecules comprise a structure represented by any oneof Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction:

-   (a) A-(B-L)J-C-M-T-D (Formula I);    -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)J-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length and comprises at least 50%        sequence complementarity to B and C; and    -   D is 0-10 nucleotides in length and comprises at least 50%        sequence complementarity to A;-   (b) D-T-T′-C-(L-B)J-A (Formula II);    -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length;    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;-   (c) T-D-M-A-(B-L)J-C (Formula III);    -   wherein T is 17-135 nucleotides in length;    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)J-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises at least 50%        sequence complementarity to D;    -   B is 4-12 nucleotides in length and comprises at least 50%        sequence complementarity to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length; or-   (d) T-D-M-A-Lp-C (Formula IV);    -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.

In some aspects, (a) T of Formula I comprises at least 80% sequencecomplementarity to B and C; (b) D of Formula I comprises at least 80%sequence complementarity to A; (c) C of Formula II comprises at least80% sequence complementarity to T; (d) B of Formula II comprises atleast 80% sequence complementarity to T; (e) A of Formula II comprisesat least 80% sequence complementarity to D; (f) A of Formula IIIcomprises at least 80% sequence complementarity to D; (g) B of FormularIII comprises at least 80% sequence complementarity to T; (h) A ofFormula IV comprises at least 80% sequence complementarity to D; and/or(i) C of Formula IV comprises at least 80% sequence complementarity toT.

In some aspects, the variant nucleic acid-guided nuclease is a Type Vvariant nucleic acid-guided nuclease. In some aspects, the one or bothof the RNP1 and the RNP2 comprise a nucleic acid-guided nucleaseselected from Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14,Cas12h, Cas12i, Cas12j, Cas13a, or Cas13b.

In some aspects of the embodiments that comprise a variant nucleicacid-guided nuclease, the variant nucleic acid-guided nuclease comprisesat least one mutation to the domains that interact with the PAM regionor surrounding sequences on the blocked nucleic acid molecules whereinthe mutation is selected from mutations to amino acid residues K538,Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acidresidues in orthologs. In some embodiments, there are at least twomutations to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules selectedfrom mutations to amino acid residues K538, Y542 and K595 in relation toSEQ ID NO:1 and equivalent amino acid residues in orthologs and in otheraspects, there are at least three mutations to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules selected from mutations to amino acid residues K538, Y542 andK595 in relation to SEQ ID NO:1 and equivalent amino acid residues inorthologs. In some aspects, the variant nucleic acid-guided nucleasecomprises at least one mutation to the domains that interact with thePAM region or surrounding sequences on the blocked nucleic acidmolecules, wherein the at least one mutation is selected from mutationsto amino acid residues K548, N552 and K607 in relation to SEQ ID NO:2;mutations to amino acid residues K534, Y538 and R591 in relation to SEQID NO:3; mutations to amino acid residues K541, N545 and K601 inrelation to SEQ ID NO:4; mutations to amino acid residues K579, N583 andK635 in relation to SEQ ID NO:5; mutations to amino acid residues K613,N617 and K671 in relation to SEQ ID NO:6; mutations to amino acidresidues K613, N617 and K671 in relation to SEQ ID NO:7; mutations toamino acid residues K617, N621 and K678 in relation to SEQ ID NO:8;mutations to amino acid residues K541, N545 and K601 in relation to SEQID NO:9; mutations to amino acid residues K569, N573 and K625 inrelation to SEQ ID NO:10; mutations to amino acid residues K562, N566and K619 in relation to SEQ ID NO:11; mutations to amino acid residuesK645, N649 and K732 in relation to SEQ ID NO:12; mutations to amino acidresidues K548, N552 and K607 in relation to SEQ ID NO:13; mutations toamino acid residues K592, N596 and K653 in relation to SEQ ID NO:14; ormutations to amino acid residues K521, N525 and K577 in relation to SEQID NO:15.

In some aspects, the variant nucleic acid-guided nuclease comprises atleast one mutation to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules, whereinsingle stranded DNA is cleaved 1.2 to 2.5 times faster than doublestranded DNA is cleaved, at least three to four times faster than doublestranded DNA is cleaved, and in some aspects, single stranded DNA iscleaved at least five times faster than double stranded DNA is cleaved.In aspects, the variant nucleic acid-guided nuclease exhibits cis- andtrans-cleavage activity.

In some aspects, the variant nucleic acid-guided nuclease comprises atleast two mutations to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules, and in someaspects, the variant nuclease comprises at least three mutations to thedomains that interact with the PAM region or surrounding sequences onthe blocked nucleic acid molecules.

In any of the embodiments comprising a concentration ratio where blockednucleic acid molecules are at a higher molar concentration than theRNP2s in the reaction mixture, certain aspects provide that theconcentration of the blocked nucleic acid molecules and the RNP2s are ata concentration ratio of at least 1.5 blocked nucleic acid molecules to1 RNP2 in the reaction mixture, and in some aspects, the concentrationof the blocked nucleic acid molecules and the RNP2s are at aconcentration ratio of at least 2 blocked nucleic acid molecules to 1RNP2 in the reaction mixture or at least 3 blocked nucleic acidmolecules to 1 RNP2, or at least 3.5 blocked nucleic acid molecules to 1RNP2, or at least 4 blocked nucleic acid molecules to 1 RNP2, or atleast 4.5 blocked nucleic acid molecules to 1 RNP2, or at least 5blocked nucleic acid molecules to 1 RNP2, or at least 5.5 blockednucleic acid molecules to 1 RNP2, or at least 6 blocked nucleic acidmolecules to 1 RNP2, or at least 6.5 blocked nucleic acid molecules to 1RNP2, or at least 7.5 blocked nucleic acid molecules to 1 RNP2, or atleast 7.5 blocked nucleic acid molecules to 1 RNP2, or at least 8blocked nucleic acid molecules to 1 RNP2, or at least 8.5 blockednucleic acid molecules to 1 RNP2, or at least 9 blocked nucleic acidmolecules to 1 RNP2, or at least 9.5 blocked nucleic acid molecules to 1RNP2, or at least 10 blocked nucleic acid molecules to 1 RNP2.

In further embodiments there is provided a variant Cas12a nucleaseengineered such that single stranded DNA is cleaved faster than doublestranded DNA is cleaved, wherein the variant Cas12a nuclease comprisesat least one mutation to the domains that interact with the PAM regionor surrounding sequences on the blocked nucleic acid molecules andwherein the variant Cas12a nuclease exhibits both cis- andtrans-cleavage activity. In some aspects, wherein the at least onemutation to the domains that interact with the PAM region or surroundingsequences on the blocked nucleic acid molecules is selected frommutations to amino acid residues K538, Y542 and K595 in relation to SEQID NO:1; the at least one mutation to the domains that interact with thePAM region or surrounding sequences on the blocked nucleic acidmolecules is selected from mutations to amino acid residues K548, N552and K607 in relation to SEQ ID NO:2; the at least one mutation to thedomains that interact with the PAM region or surrounding sequences onthe blocked nucleic acid molecules is selected from mutations to aminoacid residues K534, Y538 and R591 in relation to SEQ ID NO:3; the atleast one mutation to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules is selectedfrom mutations to amino acid residues K541, N545 and K601 in relation toSEQ ID NO:4; the at least one mutation to the domains that interact withthe PAM region or surrounding sequences on the blocked nucleic acidmolecules is selected from mutations to amino acid residues K579, N583and K635 in relation to SEQ ID NO:5; the at least one mutation to thedomains that interact with the PAM region or surrounding sequences onthe blocked nucleic acid molecules is selected from mutations to aminoacid residues K613, N617 and K671 in relation to SEQ ID NO:6; the atleast one mutation to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules is selectedfrom mutations to amino acid residues K613, N617 and K671 in relation toSEQ ID NO:7; the at least one mutation to the domains that interact withthe PAM region or surrounding sequences on the blocked nucleic acidmolecules is selected from mutations to amino acid residues K617, N621and K678 in relation to SEQ ID NO:8; the at least one mutation to thedomains that interact with the PAM region or surrounding sequences onthe blocked nucleic acid molecules is selected from mutations to aminoacid residues K541, N545 and K601 in relation to SEQ ID NO:9; the atleast one mutation to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules is selectedfrom mutations to amino acid residues K569, N573 and K625 in relation toSEQ ID NO:10; the at least one mutation to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules is selected from mutations to amino acid residues K562, N566and K619 in relation to SEQ ID NO:11; the at least one mutation to thedomains that interact with the PAM region or surrounding sequences onthe blocked nucleic acid molecules is selected from mutations to aminoacid residues K645, N649 and K732 in relation to SEQ ID NO:12; the atleast one mutation to the domains that interact with the PAM region orsurrounding sequences on the blocked nucleic acid molecules is selectedfrom mutations to amino acid residues K548, N552 and K607 in relation toSEQ ID NO:13; the at least one mutation to the domains that interactwith the PAM region or surrounding sequences on the blocked nucleic acidmolecules is selected from mutations to amino acid residues K592, N596and K653 in relation to SEQ ID NO:14; or the at least one mutation tothe domains that interact with the PAM region or surrounding sequenceson the blocked nucleic acid molecules is selected from mutations toamino acid residues K521, N525 and K577 in relation to SEQ ID NO:15including and equivalent amino acid residues in Cas12a orthologs tothese SEQ ID Nos: 1-15.

In some aspects, the variant Cas12a nuclease that has been engineeredsuch that single stranded DNA is cleaved faster than double stranded DNAis cleaved comprises any one of SEQ ID NOs: 16-600.

Alternatively, an embodiment provides a single-strand-specific Cas12anucleic acid-guided nucleases comprising an LbCas12a (i.e., SEQ IDNO: 1) with an acetylated K595 (K595K^(Ac)) residue; an AsCas12a (i.e.,SEQ ID NO: 2) with an acetylated K607 (K607K^(Ac)) residue; a CtCas12a(i.e., SEQ ID NO: 3) with an acetylated R591 (R591R^(Ac)) residue; anEeCas12a (i.e., SEQ ID NO: 4) with an acetylated K601 (K607K^(Ac))residues; an Mb3Cas12a (i.e., SEQ ID NO: 5) with an acetylated K635(K635K^(Ac)) residue; an FnCas12a (i.e., SEQ ID NO: 6) with anacetylated K671 (K671K^(Ac)) residue; an FnoCas12a (i.e., SEQ ID NO: 7)with an acetylated N671 (N671K^(Ac)) residue; an FbCas12a (i.e., SEQ IDNO: 8) with an acetylated K678 (K678K^(Ac)) residue; an Lb4Cas12a (i.e.,SEQ ID NO: 9) with an acetylated K601 (K601K^(Ac)) residue; an MbCas12a(i.e., SEQ ID NO: 10) with an acetylated K625 (K625K^(Ac)) residue; aPb2Cas12a (i.e., SEQ ID NO: 11) with an acetylated K619 (K619K^(Ac))residue; a PgCas12a (i.e., SEQ ID NO: 12) with an acetylated K732(K732K^(Ac)) residue; an AaCas12a (i.e., SEQ ID NO: 13) with anacetylated K607 (K607K^(Ac)) residue; a BoCas12a (i.e., SEQ ID NO: 14)with an acetylated K653 (K653K^(Ac)) residue; or an CmaCas12a (i.e., SEQID NO: 15) with an acetylated K577 (K577K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be a Cas12a ortholog acetylated at the amino acid of theortholog equivalent to K595 of SEQ ID NO:1.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is an overview of a prior art quantitative PCR (“qPCR”) assaywhere target nucleic acids of interest from a sample are amplifiedbefore detection.

FIG. 1B is an overview of the general principles underlying the nucleicacid-guided nuclease cascade assay described in detail herein wheretarget nucleic acids of interest from a sample do not need to beamplified before detection.

FIG. 1C is an illustration of the unwinding issue that is mitigated bythe modalities described herein.

FIG. 2A is a diagram showing the sequence of steps in an exemplarycascade assay utilizing blocked nucleic acid molecules.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid moleculeand a method for unblocking the blocked nucleic acid molecules of thedisclosure.

FIG. 2C shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula I, as described herein.

FIG. 2D shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula II, as described herein.

FIG. 2E shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula III, as described herein.

FIG. 2F shows schematics of several exemplary blocked nucleic acidmolecules containing the structure of Formula IV, as described herein.

FIG. 2G shows an exemplary single-stranded blocked nucleic acid moleculewith a design able to block R-loop formation with an RNP complex,thereby blocking activation of the trans-nuclease activity of an RNPcomplex (i.e., RNP2).

FIG. 2H shows schematics of exemplary circularized blocked nucleic acidmolecules.

FIG. 3A is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and lineartemplate molecules.

FIG. 3B is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and circulartemplate molecules.

FIG. 4 illustrates three embodiments of reporter moieties.

FIG. 5 is a simplified block diagram of an exemplary method fordesigning, synthesizing and screening variant nucleic acid-guidednucleases.

FIG. 6A shows the result of protein structure prediction using Rosettaand SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacteriumCas12a).

FIG. 6B shows the result of example mutations on the LbCas12a proteinstructure prediction using Rosetta and SWISS modeling of LbCas12a andindicating the PAM regions.

FIG. 7 is a simplified diagram of acetylating the K595 amino acid in thewildtype sequence of LbCas12a (K595K^(Ac)).

FIG. 8A is an illustration of a blocked nucleic acid molecule with bulkymodifications, cleavage thereof, and steric hindrance at thePAM-interacting (PI) domain in a nucleic acid-guided nuclease caused by5′ and 3′ modifications to a blocked nucleic acid molecule.

FIG. 8B illustrates five exemplary variations of blocked nucleic acidmolecules with bulky modifications.

FIGS. 8C, 8D and 8E list exemplary bulky modifications for 5′, 3′, andinternal positions in blocked nucleic acid molecules.

FIG. 9 is an illustration of a lateral flow assay that can be used todetect the cleavage and separation of a signal from a reporter moiety.

FIG. 10A depicts Molecule U29 and describes the properties thereof,where MU29 was used to generate the data shown in FIGS. 10B-10H.

FIG. 11A shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation G532A in thewildtype sequence.

FIG. 11B shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation K538A in thewildtype sequence.

FIG. 11C shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation Y542A in thewildtype sequence.

FIG. 11D shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation K595A in thewildtype sequence.

FIG. 11E shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutations G532A, K538A,Y5442A and K595A in the wildtype sequence.

FIG. 11F shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation K595D in thewildtype sequence.

FIG. 11G shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutation K595E in thewildtype sequence.

FIG. 11H shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutations K538A, Y542A andK595D in the wildtype sequence.

FIG. 11I shows the result of protein structure prediction using Rosettaand SWISS modeling of LbCas12a comprising the mutations K538A, Y542A andK595E in the wildtype sequence.

FIGS. 12A-12G are a series of graphs showing the time for detection ofdsDNA and ssDNA both with and without PAM sequences for wildtypeLbaCas12a and engineered variants of LbaCas12a.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DEFINITIONS

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

All of the functionalities described in connection with one embodimentof the compositions and/or methods described herein are intended to beapplicable to the additional embodiments of the compositions and/ormethods except where expressly stated or where the feature or functionis incompatible with the additional embodiments. For example, where agiven feature or function is expressly described in connection with oneembodiment but not expressly mentioned in connection with an alternativeembodiment, it should be understood that the feature or function may bedeployed, utilized, or implemented in connection with the alternativeembodiment unless the feature or function is incompatible with thealternative embodiment.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “a system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention. Conventional methodsare used for the procedures described herein, such as those provided inthe art, and demonstrated in the Examples and various generalreferences. Unless otherwise stated, nucleic acid sequences describedherein are given, when read from left to right, in the 5′ to 3′direction. Nucleic acid sequences may be provided as DNA, as RNA, or acombination of DNA and RNA (e.g., a chimeric nucleic acid).

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both limits, ranges excluding either or both of those included limitsare also included in the invention.

The term “and/or” where used herein is to be taken as specificdisclosure of each of the multiple specified features or components withor without another. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the term “about,” as applied to one or more values ofinterest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, or less in either direction (greater than or less than)of a stated reference value, unless otherwise stated or otherwiseevident from the context (except where such number would exceed 100% ofa possible value).

As used herein, the terms “binding affinity” or “dissociation constant”or “K_(d)” refer to the tendency of a molecule to bind (covalently ornon-covalently) to a different molecule. A high K_(d) (which in thecontext of the present disclosure refers to blocked nucleic acidmolecules or blocked primer molecules binding to RNP2) indicates thepresence of more unbound molecules, and a low K_(d) (which in thecontext of the present disclosure refers to unblocked nucleic acidmolecules or unblocked primer molecules binding to RNP2) indicates thepresence of more bound molecules. In the context of the presentdisclosure and the binding of blocked or unblocked nucleic acidmolecules or blocked or unblocked primer molecules to RNP2, low K_(d)values are in a range from about 100 fM to about 1 aM or lower (e.g.,100 zM) and high K_(d) values are in the range of 100 nM-100 μM (10 mM)and thus are about 10⁵- to 10¹⁰-fold or higher as compared to low K_(d)values.

As used herein, the terms “binding domain” or “binding site” refer to aregion on a protein, DNA, or RNA, to which specific molecules and/orions (ligands) may form a covalent or non-covalent bond. By way ofexample, a polynucleotide sequence present on a nucleic acid molecule(e.g., a primer binding domain) may serve as a binding domain for adifferent nucleic acid molecule (e.g., an unblocked primer nucleic acidmolecule). Characteristics of binding sites are chemical specificity, ameasure of the types of ligands that will bond, and affinity, which is ameasure of the strength of the chemical bond.

As used herein, the term “blocked nucleic acid molecule” refers tonucleic acid molecules that cannot bind to the first or second RNPcomplex to activate cis- or trans-cleavage. “Unblocked nucleic acidmolecule” refers to a formerly blocked nucleic acid molecule that canbind to the second RNP complex (RNP2) to activate trans-cleavage ofadditional blocked nucleic acid molecules. A “blocked nucleic acidmolecule” may be a “blocked primer molecule” in some embodiments of thecascade assay.

The terms “Cas RNA-guided nucleic acid-guided nuclease” or “CRISPRnuclease” or “nucleic acid-guided nuclease” refer to a CRISPR-associatedprotein that is an RNA-guided nucleic acid-guided nuclease suitable forassembly with a sequence-specific gRNA to form a ribonucleoprotein (RNP)complex.

As used herein, the terms “cis-cleavage”, “cis-nucleic acid-guidednuclease activity”, “cis-mediated nucleic acid-guided nucleaseactivity”, “cis-nuclease activity”, “cis-mediated nuclease activity”,and variations thereof refer to sequence-specific cleavage of a targetnucleic acid of interest, including an unblocked nucleic acid moleculeor synthesized activating molecule, by a nucleic acid-guided nuclease inan RNP complex. Cis-cleavage is a single turn-over cleavage event inthat only one substrate molecule is cleaved per event.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen-bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-ATCGAT-5′is 100% complementary to a region of the nucleotide sequence5′-GCTAGCTAG-3′.

As used herein, the term “contacting” refers to placement of twomoieties in direct physical association, including in solid or liquidform. Contacting can occur in vitro with isolated cells (for example ina tissue culture dish or other vessel) or in samples or in vivo byadministering an agent to a subject.

The term “conservative amino acid substitution” refers to theinterchangeability in proteins of amino acid residues having similarside chains. For example, a group of amino acids having aliphatic sidechains comprises glycine, alanine, valine, leucine, and isoleucine; agroup of amino acids having aliphatic-hydroxyl side chains comprisesserine and threonine; a group of amino acids having amide containingside chains comprises asparagine and glutamine; a group of amino acidshaving aromatic side chains comprises phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains compriseslysine, arginine, and histidine; a group of amino acids having acidicside chains comprises glutamate and aspartate; and a group of aminoacids having sulfur containing side chains comprises cysteine andmethionine. Exemplary conservative amino acid substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine-glycine, and asparagine-glutamine.

A “control” is a reference standard of a known value or range of values.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a crRNA region or guide sequence capable ofhybridizing to the target strand of a target nucleic acid of interest,and 2) a scaffold sequence capable of interacting or complexing with anucleic acid-guided nuclease. The crRNA region of the gRNA is acustomizable component that enables specificity in every nucleicacid-guided nuclease reaction. A gRNA can include any polynucleotidesequence having sufficient complementarity with a target nucleic acid ofinterest to hybridize with the target nucleic acid of interest and todirect sequence-specific binding of a ribonucleoprotein (RNP) complexcontaining the gRNA and nucleic acid-guided nuclease to the targetnucleic acid. Target nucleic acids of interest may include a protospaceradjacent motif (PAM), and, following gRNA binding, the nucleicacid-guided nuclease induces a double-stranded break either inside oroutside the protospacer region on the target nucleic acid of interest,including on an unblocked nucleic acid molecule or synthesizedactivating molecule. A gRNA may contain a spacer sequence including aplurality of bases complementary to a protospacer sequence in the targetnucleic acid. For example, a spacer can contain about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, ormore bases. The gRNA spacer may be 50%, 60%, 75%, 80%, 85%, 90%, 95%,97.5%, 98%, 99%, or more complementary to its corresponding targetnucleic acid of interest. Optimal alignment may be determined with theuse of any suitable algorithm for aligning sequences. A guide RNA may befrom about 20 nucleotides to about 300 nucleotides long. Guide RNAs maybe produced synthetically or generated from a DNA template.

“Modified” refers to a changed state or structure of a molecule.Molecules may be modified in many ways including chemically,structurally, and functionally. In one embodiment, a nucleic acidmolecule (for example, a blocked nucleic acid molecule) may be modifiedby the introduction of non-natural nucleosides, nucleotides, and/orinternucleoside linkages. In another embodiment, a modified protein(e.g., a modified or variant nucleic acid-guided nuclease) may refer toany polypeptide sequence alteration which is different from thewildtype.

The terms “percent sequence identity”, “percent identity”, or “sequenceidentity” refer to percent (%) sequence identity with respect to areference polynucleotide or polypeptide sequence following alignment bystandard techniques. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within thecapabilities of one of skill in the art, for example, using publiclyavailable computer software such as BLAST, BLAST-2, PSI-BLAST, orMegalign software. In some embodiments, the software is MUSCLE (Edgar,Nucleic Acids Res., 32(5):1792-1797 (2004)). Those skilled in the artcan determine appropriate parameters for aligning sequences, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For example, in embodiments, percentsequence identity values are generated using the sequence comparisoncomputer program BLAST (Altschul, et al., J. Mol. Biol., 215:403-410(1990)).

As used herein, the terms “preassembled ribonucleoprotein complex”,“ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complexcontaining a guide RNA (gRNA) and a nucleic acid-guided nuclease, wherethe gRNA is integrated with the nucleic acid-guided nuclease. The gRNA,which includes a sequence complementary to a target nucleic acid ofinterest, guides the RNP to the target nucleic acid of interest andhybridizes to it. The hybridized target nucleic acid-gRNA units arecleaved by the nucleic acid-guided nuclease. In the cascade assaysdescribed herein, a first ribonucleoprotein complex (RNP1) includes afirst guide RNA (gRNA) specific to a target nucleic acid of interest,and a first nucleic acid-guided nuclease, such as, for example, cas12aor cas14a for a DNA target nucleic acid, or cas13a for an RNA targetnucleic acid. A second ribonucleoprotein complex (RNP2) for signalamplification includes a second guide RNA specific to an unblockednucleic acid or synthesized activating molecule, and a second nucleicacid-guided nuclease, which may be different from or the same as thefirst nucleic acid-guided nuclease.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

As used herein, the term “sample” refers to tissues; cells or componentparts; body fluids, including but not limited to peripheral blood,serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood. “Sample”may also refer to specimens or aliquots from food; agriculturalproducts; pharmaceuticals; cosmetics, nutraceuticals; personal careproducts; environmental substances such as soil, water (from bothnatural and treatment sites), air, or sewer samples; industrial sitesand products; and chemicals and compounds. A sample further may includea homogenate, lysate or extract. A sample further refers to a medium,such as a nutrient broth or gel, which may contain cellular components,such as proteins or nucleic acid molecules.

The terms “target DNA sequence”, “target sequence”, “target nucleic acidof interest”, “target molecule of interest”, “target nucleic acid”, or“target of interest” refer to any locus that is recognized by a gRNAsequence in vitro or in vivo. The “target strand” of a target nucleicacid of interest is the strand of the double-stranded target nucleicacid that is complementary to a gRNA. The spacer sequence of a gRNA maybe 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or morecomplementary to the target nucleic acid of interest. Optimal alignmentcan be determined with the use of any suitable algorithm for aligningsequences. Full complementarity is not necessarily required providedthere is sufficient complementarity to cause hybridization andtrans-cleavage activation of an RNP complex. A target nucleic acid ofinterest can include any polynucleotide, such as DNA (ssDNA or dsDNA) orRNA polynucleotides. A target nucleic acid of interest may be located inthe nucleus or cytoplasm of a cell such as, for example, within anorganelle of a eukaryotic cell, such as a mitochondrion or achloroplast, or it can be exogenous to a host cell, such as a eukaryoticcell or a prokaryotic cell. The target nucleic acid of interest may bepresent in a sample, such as a biological or environmental sample, andit can be a viral nucleic acid molecule, a bacterial nucleic acidmolecule, a fungal nucleic acid molecule, or a polynucleotide of anotherorganism, such as a coding or a non-coding sequence, and it may includesingle-stranded or double-stranded DNA molecules, such as a cDNA orgenomic DNA, or RNA molecules, such as mRNA, tRNA, and rRNA. The targetnucleic acid of interest may be associated with a protospacer adjacentmotif (PAM) sequence, which may include a 2-5 base pair sequenceadjacent to the protospacer. In some embodiments 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more target nucleic acids can be detected by the disclosedmethod.

As used herein, the terms “trans-cleavage”, “trans-nucleic acid-guidednuclease activity”, “trans-mediated nucleic acid-guided nucleaseactivity”, “trans-nuclease activity”, “trans-mediated nuclease activity”and variations thereof refer to indiscriminate, non-sequence-specificcleavage of a target nucleic acid molecule by a nucleic acid-guidednuclease (such as by a Cas12, Cas13, and Cas14) which is triggered bybinding of N nucleotides of a target nucleic acid molecule to a gRNAand/or by cis- (sequence-specific) cleavage of a target nucleic acidmolecule. Trans-cleavage is a “multiple turn-over” event, in that morethan one substrate molecule is cleaved after initiation by a singleturn-over cis-cleavage event.

Type V CRISPR/Cas nucleic acid-guided nucleases are a subtype of Class 2CRISPR/Cas effector nucleases such as, but not limited to, engineeredCas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e),CasY (Cas12d), Cas 13a nucleases or naturally-occurring proteins, suchas a Cas12a isolated from, for example, Francisella tularensis subsp.novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (GeneID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID:15139718), and [Eubacterium] eligens ATCC 27750 (Gene ID: 41356122), andan artificial polypeptide, such as a chimeric protein.

The term “variant” in the context of the present disclosure refers to apolypeptide or polynucleotide that differs from a reference polypeptideor polynucleotide but retains essential properties. A typical variant ofa polypeptide differs in amino acid sequence from another referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many if not most regions, identical. A variant and referencepolypeptide may differ in one or more amino acid residues (e.g.,substitutions, additions, and/or deletions). A variant of a polypeptidemay be a conservatively modified variant. A substituted or insertedamino acid residue may or may not be one encoded by the genetic code(e.g., a non-natural amino acid). A variant of a polypeptide may benaturally occurring, such as an allelic variant, or it may be a variantthat is not known to occur naturally. Variants includemodifications—including chemical modifications—to one or more aminoacids that do not involve amino acid substitutions, additions ordeletions.

As used herein, the terms “variant engineered nucleic acid-guidednuclease” or “variant nucleic acid-guided nuclease” refer to nucleicacid-guided nucleases have been engineered to mutate the PAM interactingdomains in the LbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a(Acidaminococcus sp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasmatermitum Cas12a), EeCas 12a (Eubacterium eligens Cas12a), Mb3Cas12a(Moraxella bovoculi Cas12a), FnCas12a (Francisella novicida Cas12a),FnoCas12a (Francisella tularensis subsp. novicida FTG Cas12a), FbCas12a(Flavobacteriales bacterium Cas12a), Lb4Cas12a (Lachnospira eligensCas12a), MbCas12a (Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotellabryantii Cas12a), PgCas12a (Candidatus Parcubacteria bacterium Cas12a),AaCas12a (Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacteriumCas12a), CMaCas12a (Candidatus Methanomethylophilus alvus Mx1201Cas12a), and to-be-discovered equivalent Cas12a nucleic acid-guidednucleases such that double-stranded DNA (dsDNA) substrates bind to thevariant nucleic acid-guided nuclease and are cleaved by the variantnucleic acid-guided nuclease at a slower rate than single-stranded DNA(ssDNA) substrates.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like.

DETAILED DESCRIPTION

The present disclosure provides compositions of matter and methods forcascade assays that detect nucleic acids. The cascade assays allow formassive multiplexing, and provide high accuracy, low cost, minimumworkflow and results in less than one minute or, in some embodiments,virtually instantaneously, even at ambient temperatures of about 16-20°C. or less up to 48° C. The cascade assays described herein comprisefirst and second ribonucleoprotein complexes and either blocked nucleicacid molecules or blocked primer molecules. The blocked nucleic acidmolecules or blocked primer molecules keep the second ribonucleoproteincomplexes “locked” unless and until a target nucleic acid of interestactivates the first ribonucleoprotein complex. The methods comprise thesteps of providing cascade assay components, contacting the cascadeassay components with a sample, and detecting a signal that is generatedonly when a target nucleic acid of interest is present in the sample.

Early and accurate identification of, e.g., infectious agents, microbecontamination, variant nucleic acid sequences that indicate the presenceof diseases such as cancer or contamination by heterologous sources isimportant in order to select correct treatment; identify tainted food,pharmaceuticals, cosmetics and other commercial goods; and to monitorthe environment. Nucleic acid-guided nucleases, such as Type V nucleicacid-guided nucleases, can be utilized for the detection of targetnucleic acids of interest associated with diseases, food contaminationand environmental threats. However, currently available nucleic aciddetection such as quantitative PCR (also known as real time PCR or qPCR)or CRISPR-based detection assays such as SHERLOCK™ and DETECTR™ rely onDNA amplification, which requires time and may lead to changes to therelative proportion of nucleic acids, particularly in multiplexednucleic acid assays. The lack of rapidity for these detection assays isdue to the fact that there is a significant lag phase early in theamplification process where fluorescence above background cannot bedetected. With qPCR, for example, there is a lag until the cyclethreshold or Ct value, which is the number of amplification cyclesrequired for the fluorescent signal to exceed the background level offluorescence, is achieved and can be quantified.

The present disclosure describes a signal boost cascade assay andimprovements thereto that can detect one or more target nucleic acids ofinterest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower)limits in less than one minute and in some embodiments virtuallyinstantaneously without the need for amplifying the target nucleicacid(s) of interest, thereby avoiding the drawbacks of multiplexamplification, such as primer-dimerization. As described in detailbelow, the cascade assays utilize signal boost mechanisms comprisingvarious components including nucleic acid-guided nucleases, guide RNAs(gRNAs) incorporated into ribonucleoprotein complexes (RNP complexes),blocked nucleic acid molecules or blocked primer molecules, reportermoieties, and, in some embodiments, polymerases and template molecules.A particularly advantageous feature of the cascade assay is that, withthe exception of the gRNA in RNP1 (i.e., gRNA1), the cascade assaycomponents are essentially identical no matter what target nucleicacid(s) of interest are being detected, and gRNA1 is easilyprogrammable.

The improvements to the signal amplification or signal boost cascadeassay described herein result from preventing undesired unwinding of theblocked nucleic acid molecules in the reaction mix by the secondribonucleoprotein complex (RNP2) before the blocked nucleic acidmolecules are unblocked via trans-cleavage, leading to increasedefficiency, reduced background, and increased signal-to-noise ratio inthe cascade assay. Minimizing undesired unwinding serves two purposes.First, preventing undesired unwinding that happens not as a result ofunblocking due to trans-cleavage subsequent to cis-cleavage of thetarget nucleic acid of interest or trans-cleavage of unblocked nucleicacid molecules—but due to other factors—leads to a “leaky” cascade assaysystem, which in turn leads to non-specific signal generation.

Second, preventing undesired unwinding limits non-specific interactionsbetween the nucleic acid-guided nucleases (here, in the RNP2s) andblocked nucleic acid molecules such that only blocked nucleic acidmolecules that become unblocked due to trans-cleavage activity reactwith the nucleic acid-guided nucleases. This “fidelity” in the cascadeassay leads primarily to desired interactions and limits “wasteful”interactions where the nucleic acid-guided nucleases are essentiallyacting on blocked nucleic acid molecules rather than unblocked nucleicacid molecules. That is, the nucleic acid-guided nucleases are focusedon desired interactions which then leads to immediate signalamplification or boost in the cascade assay.

The present disclosure provides three modalities to minimize leakinessleading to minimal false positives or higher background signal. Thepresent disclosure demonstrates that undesired unwinding of the blockednucleic acid molecules can be lessened substantially by 1) increasingthe molar ratio of the concentration of blocked nucleic acid molecules(equivalent to a target nucleic acid molecule for the RNP2) to be equalto or greater than the molar concentration of RNP2 (e.g., the nucleicacid-guided nuclease in RNP2); 2) engineering the nucleic acid-guidednuclease used in RNP2 so as to increase the time it takes the nucleicacid-guided nuclease to recognize double-strand DNA at least two-foldand preferably three-fold or more; and/or 3) engineering the blockednucleic acid molecules to include bulky modifications (that is,molecules with a size of about 1 nm or less).

The first modality for minimizing undesired unwinding of the blockednucleic acid molecules (or blocked primer molecules) is to adjust therelative concentrations of the blocked nucleic acid molecules (orblocked primer molecules) and RNP2s such that the molar concentration ofthe blocked nucleic acid molecules (or blocked primer molecules) isequal to or greater than the molar concentration of RNP2s. Before thepresent disclosure, the common wisdom in performing CRISPR detectionassays was to use a vast excess of nucleic acid-guided nuclease (e.g.,RNP complex) to target.

In most detection assays, the quantity of the target nucleic acid ofinterest is not known (e.g., the detection assay is performed on asample with an unknown concentration of target); however, in experimentsconducted to determine the level of detection of two CRISPR detectionassays known in the art, DETECTR™ and SHERLOCK™, the nucleic acidnuclease was present at ng/μL concentrations and the target of interestwas present at very low copy numbers or at femtomolar to attamolarconcentration. Thus, the present methods and reagent mixtures not onlyadjust the relative concentrations of the blocked nucleic acid molecules(or blocked primer molecules) and RNP2s such that the molarconcentration of the blocked nucleic acid molecules (or blocked primermolecules) is equal to or greater than the molar concentration of RNP2s,but the molar concentration of RNP2s may still exceed the molarconcentration of the blocked nucleic acid molecules by a lesser amount,such as where the molar concentration of RNP2s exceeds the molarconcentration of blocked nucleic acid molecules (or blocked targetmolecules) by 100,000×, 50,000×, 25,000×, 10,000×, 5,000×, 1000×, 500×,100×, 50×, or 10× or less.

For example, Sun, et al. ran side-by-side comparisons of the DETECTR™and SHERLOCK™ detection assays, using a concentration of 100 ng/μLLbCas12a in the DETECTR™ assay and a concentration of 20 ng/μL LwCas13ain the SHERLOCK™ assay, where the concentration of the target nucleicacid molecules ranged from 0 copies/μL, 0.1 copies/μL, 0.2 copies/μL,1.0 copy/μL, 2.0 copies/μL, 5.0 copies/μL, 10.0 copies/μL, and so on upto 200.0 copies/μL. (Sun, et al., J. of Translational Medicine, 12:74(2021).) In addition, Broughton, et al., ran the DETECTR™ assay using aconcentration range of 2.5 copies/μL, to 1250 copies/μL, target nucleicacid molecules to 40 nM LbCas12 (see, Broughton, et al., Nat. Biotech.,38:870-74 (2020)); and Lee, et al., ran the SHERLOCK™ assay using aconcentration range of 10 fM to 50 aM target nucleic acid molecules to150 nM Cas12 (see Lee, et al., PNAS, 117(41):25722-31 (2020). Thus, theratio of nucleic acid-guided nuclease to blocked nucleic acid molecule(e.g., target for RNP2) described herein is very different from ratiospracticed in the art and this ratio has been determined to limitundesired unwinding of the blocked nucleic acid molecules (or blockedprimer molecules).

In a second modality, variant nucleic acid-guided nucleases have beenengineered to mutate the domains in the variants that interact with thePAM region or surrounding sequences on the blocked nucleic acidmolecules in, e.g., Type V nucleic acid-guided nucleases such as theLbCas12a (Lachnospriaceae bacterium Cas12a), AsCas 12a (Acidaminococcussp. BV3L6 Cas12a), CtCas12a (Candidatus Methanoplasma termitum Cas12a),EeCas12a (Eubacterium eligens Cas12a), Mb3Cas12a (Moraxella bovoculiCas12a), FnCas12a (Francisella novicida Cas12a), FnoCas12a (Francisellatularensis subsp. novicida FTG Cas12a), FbCas12a (Flavobacterialesbacterium Cas12a), Lb4Cas12a (Lachnospira eligens Cas12a), MbCas12a(Moraxella bovoculi Cas12a), Pb2Cas12a (Prevotella bryantii Cas12a),PgCas12a (Candidatus Parcubacteria bacterium Cas12a), AaCas12a(Acidaminococcus sp. Cas12a), BoCas12a (Bacteroidetes bacterium Cas12a),CMaCas12a (Candidatus Methanomethylophilus alvus Mx1201 Cas12a), andother related nucleic acid-guided nucleases (e.g., homologs andorthologs of these nucleic acid-guided nucleases) also limit unwinding.These variant nucleic acid-guided nucleases have been engineered suchthat double-stranded DNA (dsDNA) substrates bind to and activate to thevariant nucleic acid-guided nucleases slowly, but single-stranded DNA(ssDNA) substrates continue to bind and activate the variant nucleicacid-guided nuclease at a high rate. Thus, the variant nucleicacid-guided nucleases effect a “lock” on the RNP complex (here, theRNP2) vis-à-vis double-strand DNA. Locking RNP2 in this way lessens thelikelihood of undesired unwinding of the blocked nucleic acid moleculesas described in detail herein (see FIG. 1C and the accompanyingdiscussion). Modifying the nucleic acid-guided nucleases to notrecognize dsDNA or to recognize dsDNA is contrary to what is desired inother CRISPR-based diagnostic/detection assays.

Finally, another modality for minimizing undesired unwinding of theblocked nucleic acid molecules is to use “bulky modifications” at the 5′and/or 3′ ends of the blocked nucleic acid molecules and/or at internalnucleic acid bases of the blocked nucleic acid molecules. Doing socreates steric hindrance at the domains of the nucleic acid-guidednuclease in RNP2 that interact with the PAM region or that interact withsurrounding sequences on the blocked nucleic acid molecules, disrupting,e.g., PAM recognition in the target strand and preventing displacementof the non-target strand. Using bulky modifications is yet another pathto locking RNP2 to double-strand DNA molecules thereby lessening thelikelihood of undesired unwinding of the blocked nucleic acid moleculesas described in detail herein (again, see FIG. 1C and the accompanyingdiscussion). “Bulky modifications” include molecules with a size ofabout 1 nm or less.

FIG. 1A provides a simplified diagram demonstrating a prior art methodfor quantifying target nucleic acids of interest in a sample; namely,the quantitative polymerase chain reaction or qPCR, which to date may beconsidered the gold standard for quantitative detection assays. Thedifference between PCR and qPCR is that PCR is a qualitative techniquethat indicates the presence or absence of a target nucleic acid ofinterest in a sample, where qPCR allows for quantification of targetnucleic acids of interest in a sample. qPCR involves selectiveamplification and quantitative detection of specific regions of DNA orcDNA (i.e., the target nucleic acid of interest) using oligonucleotideprimers that flank the specific region(s) in the target nucleic acid(s)of interest. The primers are used to amplify the specific regions usinga polymerase. Like PCR, repeated cycling of the amplification processleads to an exponential increase in the number of copies of theregion(s) of interest; however, unlike traditional PCR, the increase istracked using an intercalating dye or, as shown in FIG. 1A, asequence-specific probe (e.g., a “Taq-man probe”) the fluorescence ofwhich is detected in real time. RT-qPCR differs from qPCR in that areverse transcriptase is used to first copy RNA molecules to producecDNA before the qPCR process commences.

FIG. 1A is an overview of a qPCR assay where target nucleic acids ofinterest from a sample are amplified before detection. FIG. 1A shows theqPCR method 10, comprising a double-stranded DNA template 12 and asequence specific Taq-man probe 14 comprising a region complementary tothe target nucleic acid of interest 20, a quencher 16, a quenchedfluorophore 18 where 22 denotes quenching between the quencher 16 andquenched fluorophore 18. Upon denaturation, the two strands of thedouble-stranded DNA template 12 separate into complementary singlestrands 26 and 28. In the next step, primers 24 and 24′ anneal tocomplementary single strands 26 and 28, as does the sequence-specificTaq-man probe 14 via the region complementary 20 to the complementarystrand 26 of the target nucleic acid of interest. Initially the Taq-manprobe is annealed to complementary strand 26 of the target region ofinterest intact; however, primers 24 and 24′ are extended by polymerase30 but the Taq-man probe is not, due to the absence of a 3′ hydroxygroup. Instead, the exonuclease activity of the polymerase “chews up”the Taq-man probe, thereby separating the quencher 16 from the quenchedfluorophore 18 resulting in an unquenched or excited-state fluorophore34. The fluorescence quenching ensures that fluorescence occurs onlywhen target nucleic acids of interest are present and being copied,where the fluorescent signal is proportional to the number ofsingle-strand target nucleic acids being amplified.

As noted above, the downside to the prior art, currently availabledetection assays such as qPCR, as well as CRISPR-based reaction assayssuch as SHERLOCK™ and DETECTR™ is that these assays rely on DNAamplification, which, in addition to issues with multiplexing,significantly hinders the ability to perform rapid testing, e.g., in thefield. That is, where the present cascade assay works at ambienttemperatures, including room temperatures and below, assays that requireamplification of the target nucleic acids of interest do not work wellat lower temperatures—even those assays utilizing isothermalamplification—due to non-specific binding of the primers and lowpolymerase activity. Further, primer design is far more challenging. Asfor the lack of rapidity of detection assays that require amplificationof the target nucleic acids of interest, a significant lag phase occursearly in the amplification process where fluorescence above backgroundcannot be detected, particularly in samples with very low copy numbersof the target nucleic acid of interest. And, again, amplification,particularly multiplex amplification, may cause changes to the relativeproportion of nucleic acids in samples that, in turn, lead to artifactsor inaccurate results.

FIG. 1B provides a simplified diagram demonstrating a method (100) of acascade assay. The cascade assay is initiated when the target nucleicacid of interest (104) binds to and activates a first pre-assembledribonucleoprotein complex (RNP1) (102). A ribonucleoprotein complexcomprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, wherethe gRNA is integrated with the nucleic acid-guided nuclease. The gRNA,which includes a sequence complementary to the target nucleic acid ofinterest, guides an RNP complex to the target nucleic acid of interestand hybridizes to it. Typically, preassembled RNP complexes are employedin the reaction mix—as opposed to separate nucleic acid-guided nucleasesand gRNAs—to facilitate rapid (and in the present cascade assays,virtually instantaneous) detection of the target nucleic acid(s) ofinterest.

“Activation” of RNP1 refers to activating trans-cleavage activity of thenucleic acid-guided nuclease in RNP1 (106) by binding of the targetnucleic acid-guided nuclease to the gRNA of RNP1, initiatingcis-cleavage where the target nucleic acid of interest is cleaved by thenucleic acid-guided nuclease. This binding and/or cis-cleavage activitythen initiates trans-cleavage activity (i.e., multi-turnover activity)of the nucleic acid-guided nuclease, where trans-cleavage isindiscriminate, leading to non-sequence-specific cutting of nucleic acidmolecules by the nucleic acid-guided nuclease of RNP1 (102). Thistrans-cleavage activity triggers activation of blocked ribonucleoproteincomplexes (RNP2s) (108) in various ways, which are described in detailbelow. Each newly activated RNP2 (110) activates more RNP2 (108→110),which in turn cleave reporter moieties (112). The reporter moieties(112) may be a synthetic molecule linked or conjugated to a quencher(114) and a fluorophore (116) such as, for example, a probe with a dyelabel (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end.The quencher (114) and fluorophore (116) can be about 20-30 bases apart(or about 10-11 nm apart) or less for effective quenching viafluorescence resonance energy transfer (FRET). Reporter moieties alsoare described in greater detail below.

As more RNP2s are activated (108→110), more trans-cleavage activity isactivated and more reporter moieties are activated (where here,“activated” means unquenched); thus, the binding of the target nucleicacid of interest (104) to RNP1 (102) initiates what becomes a cascade ofsignal production (120), which increases exponentially; hence, the terms“signal amplification” or “signal boost.” The cascade assay thuscomprises a single turnover event that triggers a multi-turnover eventthat then triggers another multi-turnover event in a “cascade.” Asdescribed below in relation to FIG. 4 , the reporter moieties (112) maybe provided as molecules that are separate from the other components ofthe nucleic acid-guided nuclease cascade assay, or the reporter moietiesmay be covalently or non-covalently linked to the blocked nucleic acidmolecules or synthesized activating molecules (i.e., the targetmolecules for the RNP2).

As described in detail below, the present description presents threemodalities for minimizing undesired unwinding of the blocked nucleicacid molecules (or blocked primer molecules), which possess regions ofdouble-strand DNA, where such unwinding can lead to non-specific signalgeneration and false positives. The modalities are 1) altering the ratioof the nucleic acid-guided nuclease in RNP2 to the blocked nucleic acidmolecules in contravention to the common wisdom for CRISPRdetection/diagnostic assays; 2) engineering the nucleic acid-guidednuclease used in RNP2 so that recognition of double-stranded DNA occursmore slowly than for single-strand DNA, in contravention to nucleicacid-guided nucleases that are used in other CRISPR-based detectionassays; and 3) modifying the 5′ and/or 3′ ends and/or various internalnucleic acid bases of the blocked nucleic acid molecules. One, two orall three of these modalities may be employed in a given assay.

FIG. 1C is an illustration of the effects of unwinding. FIG. 1C shows atleft a double-strand blocked nucleic acid molecule comprising a targetstrand and a non-target strand, where the non-target strand comprisesregions (shown as loops) unhybridized to the target strand. Proceedingright at top, cleavage of the loops in the non-target strand bytrans-cleavage initiated by RNP1 or RNP2 destabilizes the double-strandblocked nucleic acid molecule; that is, the now short regions of thenon-target strand that are hybridized to the target strand becomedestabilized and dehybridize. As these short regions dehybridize, thetarget strand is released and can bind to gRNA2 in RNP2, triggeringcis-cleavage of the target strand followed by trans-cleavage ofadditional blocked nucleic acid molecules. This process is the signalboost assay working as designed.

The pathway at the bottom of FIG. 1C illustrates the effect of undesiredunwinding; that is, unwinding due not to trans-cleavage as designed butby other unwinding due to recognition of the blocked nucleic acidmolecule by gRNA2 and the nucleic acid-guided nuclease in RNP2. As seenin the alternative pathway at bottom of FIG. 1C, R-loop formationbetween RNP2 and the blocked nucleic acid molecule (or blocked primermolecule) can still occur due to unwinding of the blocked nucleic acidmolecule after gRNA2 identifies the PAM. Indeed, this unwinding canoccur even in the absence of a PAM. It is an inherent characteristic ofthe biology of nucleic acid-guided nucleases.

Various components of the cascade assay, descriptions of how the cascadeassays work, and the modalities used to minimize undesired unwinding ofthe blocked nucleic acid molecules (or blocked primer molecules) aredescribed in detail below.

Target Nucleic Acids of Interest

The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule.Target nucleic acids of interest may be isolated from a sample ororganism by standard laboratory techniques or may be synthesized bystandard laboratory techniques (e.g., RT-PCR). The target nucleic acidsof interest are identified in a sample, such as a biological sample froma subject (including non-human animals or plants), items of manufacture,or an environmental sample (e.g., water or soil). Non-limiting examplesof biological samples include blood, serum, plasma, saliva, mucus, anasal swab, a buccal swab, a cell, a cell culture, and tissue. Thesource of the sample could be any mammal, such as, but not limited to, ahuman, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep, andbat. Samples may also be obtained from any other source, such as air,water, soil, surfaces, food, beverages, nutraceuticals, clinical sitesor products, industrial sites (including food processing sites) andproducts, plants and grains, cosmetics, personal care products,pharmaceuticals, medical devices, agricultural equipment and sites, andcommercial samples.

In some embodiments, the target nucleic acid of interest is from aninfectious agent (e.g., a bacteria, protozoan, insect, worm, virus, orfungus) that affects mammals, including humans. As a non-limitingexample, the target nucleic acid of interest could be one or morenucleic acid molecules from bacteria, such as Bordetella parapertussis,Bordetella pertussis, Chlamydia pneumoniae, Legionella pneumophila,Mycoplasma pneumoniae, Acinetobacter calcoaceticus-baumannii complex,Bacteroides fragilis, Enterobacter cloacae complex, Escherichia coli,Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group,Moraxella catarrhalis, Proteus spp., Salmonella enterica, Serratiamarcescens, Haemophilus influenzae, Neisseria meningitidis, Pseudomonasaeruginosa, Stenotrophomonas maltophilia, Enterococcus faecalis,Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydiatracomatis, Neisseria gonorrhoeae, Syphilis (Treponema pallidum),Ureaplasma urealyticum, Mycoplasma genitalium, and/or Gardnerellavaginalis. Also, as a non-limiting example, the target nucleic acid ofinterest could be one or more nucleic acid molecules from a virus, suchas adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E,coronavirus OC43, severe acute respiratory syndrome coronavirus 2(SARS-CoV-2), human metapneumovirus, human rhinovirus, enterovirus,influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009,influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenzavirus 3, parainfluenza virus 4, respiratory syncytial virus, herpessimplex virus 1, herpes simplex virus 2, human immunodeficiency virus(HIV), human papillomavirus, hepatitis A virus (HAV), hepatitis B virus(HBV), hepatitis C virus (HCV), and/or human parvovirus B19 (B19V).Also, as a non-limiting example, the target nucleic acid of interestcould be one or more nucleic acid molecules from a fungus, such asCandida albicans, Candida auris, Candida glabrata, Candida krusei,Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans,and/or Cryptococcus gattii. As another non-limiting example, the targetnucleic acid of interest could be one or more nucleic acid moleculesfrom a protozoan, such as Trichomonas vaginalis. See, e.g., Table 1 foran exemplary list of human pathogens, Table 2 for an exemplary list ofhuman sexually transmissible diseases.

TABLE 1 Human Pathogens NCBI Taxonomy NCBI Sequence ID Name Category IDNumber Acinetobacter baumannii Bacteria 470 GCF_008632635.1Acinetobacter calcoaceticus Bacteria 471 GCF_002055515.1 AcinetobacterBacteria 909768 Not applicable calcoaceticus-baumannii complex AnaplasmaBacteria 948 GCF_000439775.1 phagocytophilum Bacillus anthracis Bacteria1392 GCF_000008445.1 Bacteroides fragilis Bacteria 817 GCF_016889925.1Bartonella henselae Bacteria 38323 GCF_000612965.1 Bordetellaparapertussis Bacteria 519 GCF_004008295.1 Bordetella pertussis Bacteria520 GCF_004008975.1 Borrelia mayonii Bacteria 1674146 GCF_001936295.1Borrelia miyamotoi Bacteria 47466 GCF_003431845.1 Brucella abortusBacteria 235 GCF_000054005.1 Brucella melitensis Bacteria 29459GCF_000007125.1 Brucella suis Bacteria 29461 GCF_000007505.1Burkholderia mallei Bacteria 13373 GCF_002346025.1 Burkholderiapseudomallei Bacteria 28450 GCF_000756125.1 Campylobacter jejuniBacteria 197 GCF_000009085.1 Chlamydia pneumoniae Bacteria 83558GCF_000007205.1 Chlamydia psittaci Bacteria 83554 GCF_000204255.1Chlamydia Tracomatis Bacteria 813 GCF_000008725.1 Clostridium botulinumBacteria 1491 GCF_000063585.1 Clostridium perfringens Bacteria 1502GCF_020138775.1 Coxiella burnetii Bacteria 777 GCF_000007765.2 Ehrlichiachaffeesis Bacteria 945 GCF_000632965.1 Ehrlichia ewingii Bacteria 947Not available Ehrlichia ruminantium Bacteria 779 GCF_013460375.1Enterobacter cloacae Bacteria 550 GCF_000770155.1 Enterobacter cloacaeBacteria 354276 Not applicable complex Enterococcus faecalis Bacteria1351 GCF_000393015.1 Enterococcus faecium Bacteria 1352 GCF_009734005.1Escherichia coli Bacteria 562 GCF_000008865.2 Francisella tularensisBacteria 263 GCF_000156415.1 Gardnerella vaginalis Bacteria 2702GCF_002861965.1 Haemophilus influenzae Bacteria 727 GCF_000931575.1Klebsiella aerogenes Bacteria 548 GCF_007632255.1 Klebsiella oxytocaBacteria 571 GCF_003812925.1 Klebsiella pneumoniae Bacteria 573GCF_000240185.1 Legionella pneumophila Bacteria 446 GCF_001753085.1Leptospira interrogans Bacteria 173 GCF_002073495.2 Leptospirakirschneri Bacteria 29507 GCF_000243695.2 Leptospira wolffii Bacteria409998 GCF_004770635.1 Listeria monocytogenes Bacteria 1639GCF_000196035.1 Moraxella catarrhalis Bacteria 480 GCF_002080125.1Mycobacterium tuberculosis Bacteria 1773 GCF_000195955.2 Mycoplasmagenitalium Bacteria 2097 GCF_000027325.1 Mycoplasma pneumoniae Bacteria2104 GCF_900660465.1 Neisseria gonorrhoeae Bacteria 485 GCF_013030075.1Neisseria meningitidis Bacteria 487 GCF_008330805.1 Proteus hauseriBacteria 183417 GCF_004116975.1 Proteus mirabilis Bacteria 584GCF_000069965.1 Proteus penneri Bacteria 102862 GCF_022369495.1 Proteusvulgaris Bacteria 585 GCF_000754995.1 Pseudomonas aeruginosa Bacteria287 GCF_000006765.1 Rickettsia parkeri Bacteria 35792 GCF_005549115.1GCA_018610945.1 GCF_000965075.1 GCF_000965085.1 GCF_000284195.1GCF_000965145.1 Rickettsia prowazekii Bacteria 782 GCF_000277165.1Rickettsia rickettsii Bacteria 783 GCF_000017445.4 Salmonella bongoriBacteria 54736 GCF_000439255.1 Salmonella enterica Bacteria 28901GCF_000006945.2 Salmonella enterica Bacteria 28901 GCF_000006945.2Serratia marcescens Bacteria 615 GCF_003516165.1 Shigella boydiiBacteria 621 GCF_001905915.1 Shigella dysenteriae Bacteria 622GCF_001932995.2 Shigella flexneri Bacteria 623 GCF_000006925.2 Shigellasonnei Bacteria 624 GCF_013374815.1 Staphylococcus auerus Bacteria 1280GCF_000013425.1 Staphylococcus enterotoxin Bacteria 1280 U93688.2 BStaphylococcus epidermidis Bacteria 1282 GCF_006094375.1 Staphylococcuslugdunensis Bacteria 28035 GCF_001558775.1 Stenotrophomonas Bacteria40324 GCF_900475405.1 maltophilia Streptococcus agalactiae Bacteria 1311GCF_001552035.1 Streptococcus pneumoniae Bacteria 1313 GCF_002076835.1Streptococcus pyogenes Bacteria 1314 GCF_900475035.1 Treponema pallidumBacteria 160 GCF_000246755.1 Ureaplasma urealyticum Bacteria 2130GCF_000021265.1 Vibrio parahaemolyticus Bacteria 670 GCF_000196095.1Vibrio vulnificus Bacteria 672 GCF_002204915.1 Yersinia enterocoliticaBacteria 630 GCF_001160345.1 Yersinia pestis Bacteria 632GCF_000222975.1 Candida albicans Fungus 5476 GCF_000182965.3 Candidaauris Fungus 498019 GCF_002775015.1 Candida glabrata Fungus 5478GCF_000002545.3 Candida parapsilosis Fungus 5480 GCF_000182765.1 Candidatropicalis Fungus 5482 GCF_000006335.3 Coccidioides immitis Fungus 5501GCF_000149335.2 Coccidioides posadasii Fungus 199306 GCF_000151335.2Cokeromyces recurvatus Fungus 90255 GCA_000697235.1 Cryptococcus gattiiFungus 37769 GCF_000185945.1 Cryptococcus neoformans Fungus 5207GCF_000091045.1 Cunninghamella Fungus 90251 GCA_000697215.1bertholletiae Encephalitozoon cuniculi Fungus 6035 GCF_000091225.1Encephalitozoon hellem Fungus 27973 GCF_000277815.2 Encephalitozoonintestinalis Fungus 58839 GCF_000146465.1 Enterocystozoon bieneusiFungus 31281 GCF_000209485.1 Mortierella wolfii Fungus 90253GCA_016098105.1 Pichia kudriavzevii Fungus 4909 GCF_003054445.1Saksenaea vasiformis Fungus 90258 GCA_000697055.1 Syncephalastrum Fungus13706 GCA_002105135.1 racemosum Trichomonas vaginalis Fungus 5722GCF_000002825.2 Ricinus communis Plant 3988 GCF_019578655.1 Acanthamoebacastellanii Protozoa 5755 GCF_000313135.1 Babesia divergens Protozoa32595 GCA_001077455.2 Babesia microti Protozoa 5868 GCF_000691945.2Balamuthia mandrillaris Protozoa 66527 GCA_001185145.1 Cryptosporidiumparvum Protozoa 5807 GCF_000165345.1 Cyclospora cayatanensis Protozoa88456 GCF_002999335.1 Entamoeba histolytica Protozoa 5759GCF_000208925.1 Giardia lamblia Protozoa 5741 GCF_000002435.2 Naegleriafowleri Protozoa 5763 GCF_008403515.1 Toxoplasma gondii Protozoa 5811GCF_000006565.2 Alkhumra hemorrhagic Virus 172148 JF416961.1 fever virusArgentinian Virus 2169991 GCF_000856545.1 mammarenavirus Betacoronavirus1 Virus 694003 GCF_000862505.1 GCF_003972325.1 Black Creek Canal Virus1980460 GCF_002817355.1 orthohantavirus California encephalitis Virus1933264 GCF_003972565.1 orthobunyavirus Chapare mammarenavirus Virus499556 GCF_000879235.1 Chikungunya virus Virus 37124 GCF_000854045.1Crimean-Congo Virus 1980519 GCF_000854165.1 hemorrhagic feverorthnairovirus Dabie bandavirus Virus 2748958 GCF_000897355.1GCF_003087855.1 Deer tick virus Virus 58535 MZ148230 to MZ148271 Denguevirus 1 Virus 11053 GCF_000862125.1 Dengue virus 2 Virus 11060GCF_000871845.1 Dengue virus 3 Virus 11069 GCF_000866625.1 Dengue virus4 Virus 11070 GCF_000865065.1 Eastern equine encephalitis Virus 11021GCF_000862705.1 virus Enterovirus A Virus 138948 GCF_002816655.1GCF_000861905.1 GCF_001684625.1 Enterovirus B Virus 138949GCF_002816685.1 GCF_000861325.1 Enterovirus C Virus 138950GCF_000861165.1 Enterovirus D Virus 138951 GCF_000861205.1GCF_002816725.1 Guanarito mammarenavirus Virus 45219 GCF_000853765.1Heartland bandavirus Virus 2747342 GCF_000922255.1 Hendra henipavirusVirus 63330 GCF_000852685.1 Hepacivirus C Virus 11103 GCF_002820805.1GCF_000861845.1 GCF_000871165.1 GCF_000874285.1 GCF_001712785.1hepatitis A virus Virus 208726 K02990.1 M14707.1 M20273.1 X75215.1AB020564.1 hepatitis B virus Virus 10407 GCF_000861825.2 hepatitis Cvirus Virus 11103 GCF_002820805.1 GCF_000861845.1 GCF_000871165.1GCF_000874285.1 GCF_000874265.1 GCF_001712785.1 Hepatovirus A Virus12092 GCF_000860505.1 Human adenovirus A Virus 129875 GCF_000846805.1Human adenovirus B Virus 108098 GCF_000857885.1 Human adenovirus C Virus129951 GCF_000858645.1 Human adenovirus D Virus 130310 GCF_000885675.1Human adenovirus E Virus 130308 GCF_000897015.1 Human adenovirus F Virus130309 GCF_000846685.1 Human adenovirus G Virus 536079 GCF_000847325.1Human alphaherpesvirus 1 Virus 10298 GCF_000859985.2 Humanalphaherpesvirus 2 Virus 10310 GCF_000858385.2 human betaherpesvirus 6AVirus 32603 GCF_000845685.2 human betaherpesvirus 6B Virus 32604GCF_000846365.1 Human coronavirus 229E Virus 11137 GCF_001500975.1GCF_000853505.1 Human coronavirus HKU1 Virus 290028 GCF_000858765.1Human coronavirus NL63 Virus 277944 GCF_000853865.1 Human coronavirusOC43 Virus 31631 GCF_003972325.1 Human gammaherpesvirus Virus 37296GCF_000838265.1 8 Human immunodeficiency Virus 11676 GCF_000864765.1virus 1 Human immunodeficiency Virus 11709 GCF_000856385.1 virus 2 humanmetapneumovirus Virus 162145 GCF_002815375.1 human papillomavirus VirusGCF_001274345.1 Human polyomavirus 1 Virus 1891762 GCF_000837865.1 Humanpolyomavirus 2 Virus 1891763 GCF_000863805.1 human rhinovirus A Virus147711 GCF_000862245.1 GCF_002816835.1 human rhinovirus B Virus 147712GCF_000861265.1 GCF_002816855.1 human rhinovirus C Virus 463676GCF_002816885.1 GCF_000872325.1 Influenza A virus Virus 11320GCF_001343785.1 GCF_000851145.1 GCF_000866645.1 Influenza B virus Virus11520 GCF_000820495.2 Influenza C virus Virus 11552 GCF_000856665.10Influenza D virus Virus 1511084 GCF_002867775.1 Japanese encephalitisvirus Virus 11072 GCF_000862145.1 Kyasanur Forest disease Virus 33743GCF_002820625.1 virus La Crosse orthobunyavirus Virus 2560547GCF_000850965.1 Lassa virus Virus 11620 GCF_000851705.1 Lujomammarenavirus Virus 649188 GCF_000885555.1 Lyssavirus australis Virus90961 GCF_000850325.1 Marburg virus Virus NC_001608.3 Measlesmorbillivirus Virus 11234 GCF_000854845.1 Middle East respiratory Virus1335626 GCF_002816195.1 syndrome-related GCF_000901155.1 coronavirusMonongahela hantavirus Virus 2259728 MH539865 MH539866 MH539867 New Yorkhantavirus Virus 44755 U36803.1 U36802.1 U36801.1 U09488.1 Nipahhenipavirus Virus 121791 GCF_000863625.1 Norwalk virus Virus 11983GCF_000864005.1 GCF_008703965.1 GCF_008703985.1 GCF_008704025.1GCF_010478905.1 GCF_000868425.1 Omsk hemorrhagic fever Virus 12542GCF_000855505.1 virus parainfluenza virus 1 Virus 12730 GCF_000848705.1NC_003461 parainfluenza virus 2 Virus X57559.1 AF533010 AF533011AF533012 parainfluenza virus 3 Virus 11216 GCA_006298365.1GCA_000850205.1 parainfluenza virus 4 Virus 2560526 NC_021928.1Paslahepevirus balayani Virus 1678141 GCF_000861105.1 Poliovirus Virus138950 GCF_000861165.1 Primate erythroparvovirus 1 Virus 1511900GCF_000839645.1 Rabies lyssavirus Virus 11292 GCF_000859625.1respiratory syncytial virus Virus 12814 GCF_000856445.1 Rift Valleyvirus Virus 11588 HE687302 HE687307 Saint Louis encephalitis Virus 11080GCF_000866785.1 virus GCF_000849945.1 GCF_000855765.1 Sapporo virusVirus 95342 GCF_000854265.1 GCF_001008475.1 GCF_000853825.1 SARS-relatedcoronavirus Virus 694009 GCF_000864885.1 GCF_009858895.2 Severe acuterespiratory Virus 2901879 NC_004718.3 syndrome coronavirus 1 Severeacute respiratory Virus 2697049 NC_045512.2 syndrome coronavirus 2 SinNombre virus Virus 1980491 GCF_000854765.1 Tick-borne encephalitis Virus11084 GCF_000863125.1 virus Variola major Virus 12870 not availableVariola minor Virus 53258 not available Variola virus Virus 10255GCF_000859885.1 Venezuelan equine Virus 11036 GCF_000862105.1encephalitis virus West Nile virus Virus 11082 GCF_000861085.1GCF_000875385.1 Western equine encephalitis Virus 11039 GCF_000850885.1virus Yellow fever virus Virus 11089 GCF_000857725.1 Zaire ebolavirusVirus 186538 GCF_000848505.1 Zika virus Virus 64320 GCF_000882815.3GCF_002366285.1

TABLE 2 Human STD pathogens NCBI Taxonomy NCBI Sequence Name Category IDID Number Pthirus pubis Animal 121228 MT721740.1 Sarcoptes scabieiAnimal 52283 GCA_020844145.1 Chlamydia trachomatis Bacteria 813GCF_000008725.1 Gardnerella vaginalis Bacteria 2702 GCF_002861965.1Haemophilus ducreyi Bacteria 730 GCF_001647695.1 Mycoplasma genitaliumBacteria 2097 GCF_000027325.1 Neisseria gonorrhoeae Bacteria 485GCF_013030075.1 Treponema pallidum Bacteria 160 GCF_000246755.1Trichomonas vaginalis Protozoa 5722 GCF_000002825.2 Hepacivirus C Virus11103 GCF_002820805.1 Hepatitis B virus Virus 10407 GCF_000861825.2Hepatitis delta virus Virus 12475 GCF_000856565.1 Hepatovirus A Virus12092 GCF_000860505.1 Human alphaherpesvirus 1 Virus 10298GCF_000859985.2 Human immunodeficiency Virus 11676 GCF_000864765.1 virus1 Human immunodeficiency Virus 11709 GCF_000856385.1 virus 2 Humanpapillomavirus Virus 10566 GCF_001274345.1

Additionally, the target nucleic acid of interest may originate in anorganism such as a bacterium, virus, fungus or other pest that infectslivestock or agricultural crops. Such organisms include avian influenzaviruses, mycoplasma and other bovine mastitis pathogens, Clostridiumperfringens, Campylobacter sp., Salmonella sp., Pospirivoidae,Avsunvirodiae, Panteoea stewartii, Mycoplasma genitalium, Sprioplasmasp., Pseudomonas solanacearum, Erwinia amylovora, Erwinia carotovora,Pseudomonas syringae, Xanthomonas campestris, Agrobacterium tumefaciens,Spiroplasma citri, Phytophthora infestans, Endothia parasitica,Ceratocysis ulmi, Puccinia graminis, Hemilea vastatrix, Ustilage maydis,Ustilage nuda, Guignardia bidwellii, Uncinula necator, Botrytiscincerea, Plasmopara viticola, or Botryotinis fuckleina. See, e.g.,Table 3 for an exemplary list of non-human animal pathogens.

TABLE 3 Animal Pathogens NCBI Taxonomy NCBI Sequence Name Category ID IDNumber Acarapis woodi Animal 478375 GCA_023170135.1 Aethina tumidaAnimal 116153 GCF_001937115.1 Chorioptes bovis Animal 420257 Chrysomyabezziana Animal 69364 Cochliomyia hominivorax Animal 115425GCA_004302925.1 Echinococcus granulosus Animal 6210 GCF_000524195.1Echinococcus Animal 6211 GCA_000469725.3 multilocularis Gyrodactylussalaris Animal 37629 GCA_000715275.1 Psoroptes ovis Animal 83912GCA_002943765.1 Sarcoptes scabiei Animal 52283 GCA_020844145.1 Taeniasolium Animal 6204 GCA_001870725.1 Trichinella britovi Animal 45882GCA_001447585.1 Trichinella nativa Animal 6335 GCA_001447565.1Trichinella nelsoni Animal 6336 GCA_001447455.1 Trichinella papuaeAnimal 268474 GCA_001447755.1 Trichinella pseudospiralis Animal 6337GCA_001447645.1 Trichinella spiralis Animal 6334 GCF_000181795.1Trichinella zimbabwensis Animal 268475 GCA_001447665.1 Tropilaelapsclareae Animal 208209 Tropilaelaps koenigerum Animal 208208 Tropilaelapsmercedesae Animal 418985 GCA_002081605.1 Tropilaelaps thaii Animal418986 Varroa destructor Animal 109461 GCF_002443255.1 Varroa jacobsoniAnimal 62625 GCF_002532875.1 Varroa rindereri Animal 109259 Varroaunderwoodi Animal 109260 Anaplasma centrale Bacteria 769 GCF_000024505.1Anaplasma marginale Bacteria 770 GCF_000020305.1 Bacillus anthracisBacteria 1392 GCF_000008445.1 Brucella abortus Bacteria 235GCF_000054005.1 Brucella melitensis Bacteria 29459 GCF_000007125.1Brucella ovis Bacteria 236 GCF_000016845.1 Brucella suis Bacteria 29461GCF_000007505.1 Burkholderia mallei Bacteria 13373 GCF_002346025.1Burkholderia pseudomallei Bacteria 28450 GCF_000756125.1 Campylobacterfetus Bacteria 196 GCF_000015085.1 Candidatus Xenohaliotis Bacteria84677 californiensis Candidatus Hepatobacter Bacteria 1274402GCF_000742475.1 penaei Chlamydia abortus Bacteria 83555 GCF_900416725.2Chlamydia psittaci Bacteria 83554 GCF_000204255.1 CorynebacteriumBacteria 1719 GCF_001865765.1 pseudotuberculosis Coxiella burnetiiBacteria 777 GCF_000007765.2 Ehrlichia ruminantium Bacteria 779GCF_013460375.1 Francisella tularensis Bacteria 263 GCF_000156415.1Melissococcus plutonius Bacteria 33970 GCF_003966875.1 Mycobacteriumavium Bacteria 1764 GCF_000696715.1 Mycobacterium Bacteria 1773GCF_000195955.2 tuberculosis Mycoplasma capricolum Bacteria 2095GCF_000012765.1 Mycoplasma gallisepticum Bacteria 2096 GCF_000286675.1Mycoplasma mycoides Bacteria 2102 GCF_000023685.1 Mycoplasmaputrefaciens Bacteria 2123 GCF_900476175.1 Mycoplasmopsis agalactiaeBacteria 2110 GCF_009150585.1 Mycoplasmopsis synoviae Bacteria 2109GCF_013393745.1 Paenibacillus larvae Bacteria 1464 GCF_002951935.1Pasteurella multocida Bacteria 747 GCF_000006825.1 Salmonella entericaBacteria 28901 GCF_000006945.2 Streptococcus equi Bacteria 1336GCF_015689455.1 Taylorella equigenitalis Bacteria 29575 GCF_002288025.1Vibrio parahaemolyticus Bacteria 670 GCF_000196095.1 Batrachochy triumFungi 109871 GCF_000203795.1 dendrobatidis Batrachochy trium Fungi1357716 GCA_021556675.1 salamandrivorans Aphanomyces astaci Oomycota112090 GCF_000520075.1 Aphanomyces invadans Oomycota 157072GCF_000520115.1 Babesia bigemina Protozoa 5866 GCF_000981445.1 Babesiabovis Protozoa 5865 GCA_000165395.2 Babesia caballi Protozoa 5871Bonamia exitiosa Protozoa 362532 Bonamia ostreae Protozoa 126728Leishmania amazonensis Protozoa 5659 GCA_005317125.1 Leishmaniabraziliensis Protozoa 5660 GCF_000002845.2 Leishmania donovani Protozoa5661 GCF_000227135.1 Leishmania infantum Protozoa 5671 GCF_000002875.2Leishmania major Protozoa 5664 GCF_000002725.2 Leishmania mexicanaProtozoa 5665 GCF_000234665.1 Leishmania tropica Protozoa 5666GCA_014139745.1 Marteilia refringens Protozoa 107386 Perkinsus marinusProtozoa 31276 GCF_000006405.1 Perkinsus olseni Protozoa 32597GCA_013115135.1 Theileria annulata Protozoa 5874 GCF_000003225.4Theileria equi Protozoa 5872 GCF_000342415.1 Theileria parva Protozoa5875 GCF_000165365.1 Tritrichomonas foetus Protozoa 1144522GCA_001839685.1 Trypanosoma brucei Protozoa 5691 GCF_000002445.2Trypanosoma congolense Protozoa 5692 GCA_002287245.1 Trypanosomaequiperdum Protozoa 5694 GCA_001457755.2 Trypanosoma evansi Protozoa5697 GCA_917563935.1 Trypanosoma vivax Protozoa 5699 GCA_021307395.1African horse Virus 40050 GCF_000856125.1 sickness virus African swinefever virus Virus 10497 GCF_000858485.1 Akabane orthobunyavirus Virus1933178 GCF_000871205.1 Alcelaphine Virus 35252 GCF_000838825.1gammaherpesvirus 1 Alphaarterivirus equid Virus 2499620 GCF_000860865.1Alphacoronavirus 1 Virus 693997 GCF_000856025.1 Ambystoma tigrinum virusVirus 265294 GCF_000841005.1 Avian coronavirus Virus 694014GCF_012271565.1 Avian influenza virus Virus 11309 Avian metapneumovirusVirus 38525 GCF_002989735.1 Avian orthoavulavirus 1 Virus 2560319GCF_002834085.1 Avihepatovirus A Virus 691956 GCF_000869945.1Betaarterivirus suid 1 Virus 2499680 GCF_003971765.1 Bluetongue virusVirus 40051 GCF_000854445.3 Bovine alphaherpesvirus 1 Virus 10320GCF_008777455.1 Bovine leukemia virus Virus 11901 GCF_000853665.1Camelpox virus Virus 28873 GCF_000839105.1 Caprine arthritis Virus 11660GCF_000857525.1 encephalitis virus Crimean-Congo Virus 1980519GCF_000854165.1 hemorrhagic fever orthonairovirus Cyprinid herpesvirus 3Virus 180230 GCF_000871465.1 Decapod iridescent virus 1 Virus 2560405GCF_00478 8555.1 Decapod Virus 1513224 GCF_000844705.1 penstyldensovirus1 Deformed wing virus Virus 198112 GCF_000852585.1 Eastern equine Virus11021 GCF_000862705.1 encephalitis virus Epizootic haematopoietic Virus100217 GCF_001448375.1 necrosis virus Epizootic hemorrhagic Virus 40054GCF_000885335.1 disease virus Equid alphaherpesvirus 1 Virus 10326GCF_000844025.1 Equid alphaherpesvirus 4 Virus 10331 GCF_000846345.1Equine infectious Virus 11665 GCF_000847605.1 anemia virusFoot-and-mouth disease Virus 12110 GCF_002816555.1 virus Frog virus 3Virus 10493 GCF_002826565.1 Gallid alphaherpesvirus 1 Virus 10386GCF_000847005.1 Goatpox virus Virus 186805 GCF_000840165.1 Haliotidherpesvirus 1 Virus 1513231 GCF_000900375.1 Hendra henipavirus Virus63330 GCF_000852685.1 Infectious bursal Virus 10995 GCF_000855485.1disease virus Infectious spleen Virus 180170 GCF_000848865.1 and kidneynecrosis virus Influenza A virus Virus 11320 GCF_000851145.1 Isavirussalaris Virus 55987 GCF_000854145.2 Japanese encephalitis virus Virus11072 GCF_000862145.1 Lumpy skin disease virus Virus 59509GCF_000839805.1 Lyssavirus rabies Virus 11292 GCF_000859625.1Macrobrachium Virus 222557 GCA_000856985.1 rosenbergii nodavirus MiddleEast respiratory Virus 1335626 GCF_002816195.1 syndrome-relatedcoronavirus Myxoma virus Virus 10273 GCF_000843685.1 Nairobi sheep Virus1980526 GCF_002117695.1 disease orthonairovirus Nipah henipavirus Virus121791 GCF_000863625.1 Norwegian salmonid Virus 344701 alphavirusNovirhabdovirus piscine Virus 1980916 GCF_000856505.1 Novirhabdovirussalmonid Virus 1980917 GCF_000850065.1 Penaeid shrimp infectious Virus282786 GCA_000866305.1 myonecrosis virus Peste des petits ruminantsVirus 2593991 GCF_000866445.1 virus Pestivirus C Virus 2170082GCF_000864685.1 GCF_003034095.1 Pestivirus A Virus 2170080GCF_000861245.1 Rabbit hemorrhagic Virus 11976 GCF_000861285.1 diseasevirus Rift Valley fever Virus 1933187 GCF_000847345.1 phlebovirusRinderpest morbillivirus Virus 11241 GCF_000856645.1 Severe acute Virus694009 GCF_000864885.1 respiratory syndrome- related coronavirusSheeppox virus Virus 10266 GCF_000840205.1 Slow bee paralysis virusVirus 458132 GCF_0008 87395.1 Sprivirus cyprinus Virus 696863GCF_000850305.1 Suid alphaherpesvirus 1 Virus 10345 GCF_000843 825.1Swine vesicular Virus 12075 disease virus Taura syndrome virus Virus142102 GCF_000849385.1 Tilapinevirus tilapiae Virus 2034996GCF_001630085.1 Venezuelan equine Virus 11036 GCF_000862105.1encephalitis virus Vesiculovirus Indiana Virus 1972577 GCF_000850045.1Visna-maedi virus Virus 2169971 GCF_000849025.1 West Nile Virus Virus11082 GCF_000861085.1 Western equine Virus 11039 GCF_OOO85O885.1encephalitis virus White spot syndrome virus Virus 342409GCF_000848085.2 Yellow head virus Virus 96029 GCF_003972805.1

In some embodiments, other target nucleic acids of interest may be fornon-infectious conditions, e.g., to be used for genotyping, includingnon-invasive prenatal diagnosis of, e.g, trisomies, other chromosomalabnormalities, and known genetic diseases such as Tay Sachs disease andsickle cell anemia. Other target nucleic acids of interest and samplesare described herein, such as human biomarkers for cancer. An exemplarylist of human biomarkers is in Table 4. Target nucleic acids of interestmay include engineered biologics, including cells such as CAR-T cells,or target nucleic acids of interest from very small or rare samples,where only small volumes are available for testing.

TABLE 4 Human Biomarkers NCBI NCBI Taxonomy Gene Biomarker DiseaseSample ID ID Aβ42, amyloid beta- Alzheimer disease CSF 9606 351 proteinprion protein Alzheimer disease, CSF 9606 5621 prion disease Vitamin Dbinding multiple sclerosis CSF 9606 2638 protein progression CXCL13multiple sclerosis CSF 9606 10563 alpha-synuclein parkinsonian disordersCSF 9606 6622 tau protein parkinsonian disorders CSF 9606 4137 Apo IIparkinsonian disorders CSF 9606 336 ceruloplasmin parkinsonian disordersCSF 9606 1356 peroxisome parkinsonian disorders CSF 9606 5467proliferation- activated PD receptor parkin neurogenerative CSF 96065071 disorders PTEN induced neurogenerative CSF 9606 65018 putativekinase I disorders DJ-1 (PARK7) neurogenerative CSF 9606 11315 disordersleucine-rich repeat neurogenerative CSF 9606 120892 kinase disorderssecretogranin II bipolar disorder CSF 9606 7857 neurofilament lightaxonal degeneration CSF 9606 4747 chain IL-12B, CXDL13, Intrathecal CSF9606 3593, 10563, IL-8 inflammation 3576 ACE2 cardiovascular diseaseblood 9606 59272 alpha-amylase cardiovascular disease saliva 9606 276alpha-feto protein pregnancy blood 9606 174 albumin urine diabetes 9606213 albumin, urea albuminuria urine 9606 213 neutrophil gelatinase-acute kidney injury urine 9606 3934 associated lipocalin (NGAL) IL-18acute kidney injury urine 9606 3606 liver fatty acid acute kidney injuryurine 9606 2168 binding protein Dkk-3 prostate cancer semen 9606 27122autoantibody to early diagnosis blood 9606 CD25 esophageal squamous cellcarcinoma hTERT lung cancer blood 9606 7015 CAI 25 (MUC16) lung cancerblood 9606 94025 VEGF lung cancer blood 9606 7422 IL-2 lung cancer blood9606 3558 osteopontin lung cancer blood 9606 6696 BRAF, CCNI, EGRF, lungcancer saliva 9606 673, 16007, FGF19, FRS2, 1956, 9965, GREB1, and LZTS110818, 9687, 11178 human epididymis ovarian cancer blood 9606 10406protein 4 CA125 ovarian cancer saliva 9606 94025 EMP1 nasopharyngealsaliva 9606 13730 carcinoma IL-8 oral cancer saliva 9606 3576carcinoembryonic oral or salivary saliva 9606 1048 antigen malignanttumors thioredoxin Spinalcellular carcinoma saliva 9606 7295 AIP (arylAcute intermittent blood 9606 9049 hydrocarbon receptor porphyria,somatotroph interacting protein) adenoma, prolactin- producing pituitarygland adenoma ALK receptor Neuroblastoma blood 9606 238 tyrosine kinasesusceptibility, large cell lymphoma BAP1 (BRCA1 BAP1-related tumor blood9606 8314 associated protein 1) predisposition, melanoma susceptibilityBLM Bloom syndrome blood 9606 641 BRCA1 Breast-ovarian cancer blood 9606672 susceptibility, familial breast cancer BRCA2 Breast-ovarian cancerblood 9606 675 susceptibility, familial breast cancer, gliomasusceptibility CASR (calcium Epilepsy susceptibility blood 9606 846sensing receptor) CDC73 Hyperparathyroidism 2 blood 9606 79577 with jawtumors CEBPA Acute myloid leukemia blood 9606 1050 EPCAM Colorectalcancer blood 9606 4072 FH hypercholesterolemia blood 9606 2271 GATA2Acute myeloid leukemia blood 9606 2642 MITF Melanoma susceptibilityblood 9606 4286 MSH2 Lynch syndrome blood 9606 4436 MSH3 Endometrialcarcinoma blood 9606 4437 MSH6 Endometrial carcinoma, blood 9606 2956colorectal cancer NF1 Neurofibromatosis, blood 9606 4763 juvenilemyelomonocytic leukemia PDGRA Eosinophilic leukemia, blood 9606 5156recurrent inflammatory gastrointestinal fibroids PHOX2B Neuroblastomablood 9606 8929 susceptibility POTI Melanoma blood 9606 25913susceptibility, glioma susceptibility

The target nucleic acids of interest may be taken from environmentalsamples. A list of exemplary biosafety pathogens is in Table 5, and anexemplary list of known viruses is in Table 6.

TABLE 5 Exemplary Laboratory Biosafety Parasites and Pathogens NCBITaxonomy Name Category ID Acarapis woodi Animal 478375 Aethina tumidaAnimal 116153 Alaria americana Animal 2282137 Amblyomma Animal 6943americanum Amblyomma maculatum Animal 34609 Amphimerus Animalpseudofelineus Ancylostoma braziliense Animal 369059 Ancylostoma caninumAnimal 29170 Ancylostoma duodenale Animal 51022 Anisakis pegreffiiAnimal 303229 Anisakis simplex Animal 6269 Baylisascaris columnarisAnimal 575210 Baylisascaris melis Animal Baylisascaris procyonis Animal6259 Bunostomum Animal 577651 phlebotomum Ceratonova shasta Animal 60662Chrysomya bezziana Animal 69364 Cochliomyia Animal 115425 hominivoraxDicrocoelium Animal 57078 dendriticum Diphyllobothrium Animal 28845dendriticum Diphyllobothrium latum Animal 60516 Echinococcus granulosaAnimal Echinococcus multilocularis Animal 6211 Echinococcus oligarthrusAnimal 6212 Echinococcus shiquicus Animal 260967 Echinococcus vogeliAnimal 6213 Echinostoma cinetorchis Animal 1873862 Echinostoma hortenseAnimal 48216 Echinostoma liei Animal 48214 Echinostoma revolutum Animal48217 Fasciola hepatica Animal 6192 Fascioloides magna Animal 394415Gyrodactylus salaris Animal 37629 Ixodes pacificus Animal 29930 Ixodesricinus Animal 34613 Ixodes scapularis Animal 6945 Metagonimus yokogawaiAnimal 84529 Metorchis conjunctus Animal Myxobolus cerebralis Animal59783 Nanophyetuss almincola Animal 240278 Necator americanus Animal51031 Oestrus ovis Animal 123737 Opisthorchis felineus Animal 147828Opisthorchis viverrini Animal 6198 Parafilaria bovicola Animal 2282233Paragonimus kellicotti Animal 100269 Paragonimus miyazakii. Animal 59628Paragonimus Animal 34504 westermani Psoroptes ovis Animal 83912Rhipicephalus annulatus Animal 34611 Rhipicephalus sanguineus Animal34632 Sarcoptes scabiei Animal 52283 Taenia multiceps Animal 94034Taenia saginata Animal 6206 Taenia solium Animal 6204 Toxocara canisAnimal 6265 Toxocara cati Animal 6266 Trichinella spiralis Animal 6334Trichuris suis Animal 68888 Trichuris trichiura Animal 36087 Trichurisvulpis Animal 219738 Tropilaelaps clareae Animal 208209 Tropilaelapsmercedesae Animal 418985 Uncinaria stenocephala Animal 125367 Varroadestructor Animal 109461 Actinobacillus Bacteria 715 pleuropneumoniaeAeromonas hydrophila Bacteria 644 Aeromonas salmonicida Bacteria 645Aliarcobacter butzleri Bacteria 28197 Aliarcobacter Bacteria 28198cryaerophilus Aliarcobacter skirrowii Bacteria 28200 Anaplasma centraleBacteria 769 Anaplasma marginale Bacteria 770 Anaplasma Bacteria 948phagocytophilum Bacillus anthracis Bacteria 1392 Bacillus cereusBacteria 1396 Bartonella henselae Bacteria 38323 Bibersteinia trehalosiBacteria 47735 Borrelia burgdorferi Bacteria 139 Brucella abortusBacteria 235 Brucella canis Bacteria 36855 Brucella melitensis Bacteria29459 Brucella ovis Bacteria 236 Brucella suis Bacteria 29461Burkholderia mallei Bacteria 13373 Burkholderia Bacteria 28450pseudomallei Campylobacter coli Bacteria 195 Campylobacter fetus fetusBacteria 32019 Campylobacter fetus Bacteria 32020 venerealisCampylobacter jejuni Bacteria 197 Chlamydia caviae Bacteria 83557Chlamydia felis Bacteria 83556 Chlamydia muridarum Bacteria 83560Chlamydia pecorum Bacteria 85991 Chlamydia pneumoniae Bacteria 83558Chlamydia psittaci Bacteria 83554 Chlamydia suis Bacteria 83559Chlamydia trachomatis Bacteria 813 Chlamydophilus abortus BacteriaClostridium botulinum Bacteria 1491 Clostridium difficile Bacteria 1496Clostridium perfringens Bacteria Types A, B, C, and D Coxiella burnetiiBacteria 777 Cronobacter sakazakii Bacteria 28141 Ehrlichia canisBacteria 944 Ehrlichia chaffeensis Bacteria 945 Ehrlichia ewingiiBacteria 947 Ehrlichia ondiri Bacteria Ehrlichia ruminantium Bacteria779 Escherichia coli Bacteria 562 Klebsiella aerogenes Bacteria 548Klebsiella granulomatis Bacteria 39824 Klebsiella grimontii Bacteria2058152 Klebsiella huaxiensis Bacteria 2153354 Klebsiella kielensisBacteria 2042302 Klebsiella michiganensis Bacteria 1134687 Klebsiellamilletis Bacteria 223378 Klebsiella oxytoca Bacteria 571 Klebsiellapneumoniae Bacteria 573 Klebsiella quasipneumoniae Bacteria 1463165Klebsiella quasivariicola Bacteria 2026240 Klebsiella senegalensisBacteria 223379 Klebsiella steroids Bacteria 1641362 Klebsiellavariicola Bacteria 244366 Proteus mirabilis Bacteria 584 Pseudomonasabietaniphila Bacteria 89065 Pseudomonas acephalitica Bacteria 407029Pseudomonas acidophila Bacteria 1912599 Pseudomonas adelgestsugasBacteria 1302376 Pseudomonas aeruginosa Bacteria 287 Pseudomonas aestusBacteria 1387231 Pseudomonas agarici Bacteria 46677 Pseudomonasakappageensis Bacteria Pseudomonas alcaligenes Bacteria 43263Pseudomonas alcaliphila Bacteria 101564 Pseudomonas alginovora Bacteria37638 Pseudomonas alkanolytica Bacteria Pseudomonas Bacteria 237609alkylphenolica Pseudomonas allii Bacteria 2740531 Pseudomonasalliivorans Bacteria 2810613 Pseudomonas Bacteria 2774460 allokribbensisPseudomonas alloputida Bacteria 1940621 Pseudomonas alvandae Bacteria2842348 Pseudomonas amygdali Bacteria 47877 Pseudomonas Bacteria 32043amyloderamosa Pseudomonas anatoliensis Bacteria 2710589 Pseudomonasandersonii Bacteria 147728 Pseudomonas Bacteria 53406 anguillisepticaPseudomonas antarctica Bacteria 219572 Pseudomonas Bacteria 485870anuradhapurensis Pseudomonas Bacteria 2710591 arcuscaelestis PseudomonasBacteria 289370 argentinensis Pseudomonas Bacteria 702115 arsenicoxydansPseudomonas Bacteria 2842349 asgharzadehiana Pseudomonas asiaticaBacteria 2219225 Pseudomonas asplenii Bacteria 53407 Pseudomonasasturiensis Bacteria 1190415 Pseudomonas asuensis Bacteria 1825787Pseudomonas atacamensis Bacteria 2565368 Pseudomonas atagonensisBacteria 2609964 Pseudomonas aurantiaca Bacteria 86192 Pseudomonasaureofaciens Bacteria 587851 Pseudomonas avellanae Bacteria 46257Pseudomonas Bacteria 1869229 aylmerensis Pseudomonas azadiae Bacteria2843612 Pseudomonas Bacteria azerbaij anoccidentalis PseudomonasBacteria azerbaij anorientalis Pseudomonas azotifigens Bacteria 291995Pseudomonas Bacteria 47878 azotoformans Pseudomonas baetica Bacteria674054 Pseudomonas balearica Bacteria 74829 Pseudomonas baltica Bacteria2762576 Pseudomonas Bacteria 2843610 bananamidigenes Pseudomonasbathycetes Bacteria Pseudomonas batumici Bacteria 226910 PseudomonasBacteria 556533 benzenivorans Pseudomonas bijieensis Bacteria 2681983Pseudomonas Bacteria 254015 blatchfordae Pseudomonas bohemica Bacteria2044872 Pseudomonas borbori Bacteria 289003 Pseudomonas borealisBacteria 84586 Pseudomonas botevensis Bacteria 2842352 PseudomonasBacteria 930166 brassicacearum Pseudomonas Bacteria 2708063 brassicaePseudomonas brenneri Bacteria 129817 Pseudomonas bubulae Bacteria2316085 Pseudomonas campi Bacteria 2731681 Pseudomonas canadensisBacteria 915099 Pseudomonas Bacteria 2859001 canavaninivoransPseudomonas cannabina Bacteria 86840 Pseudomonas capeferrum Bacteria1495066 Pseudomonas capsici Bacteria 2810614 Pseudomonas Bacteria 46678caricapapayae Pseudomonas carnis Bacteria 2487355 Pseudomonas caspianaBacteria 1451454 Pseudomonas cavernae Bacteria 2320867 PseudomonasBacteria 2320866 cavernicola Pseudomonas cedrina Bacteria 651740Pseudomonas cellulosa Bacteria 155077 Pseudomonas cerasi Bacteria1583341 Pseudomonas chaetocerotis Bacteria Pseudomonas chengduensisBacteria 489632 Pseudomonas Bacteria 203192 chloritidismutansPseudomonas chlororaphis Bacteria 587753 Pseudomonas cichorii Bacteria36746 Pseudomonas citronellolis Bacteria 53408 Pseudomonas clemanceaBacteria 416340 Pseudomonas coenobios Bacteria Pseudomonas Bacteria1605838 coleopterorum Pseudomonas composti Bacteria 658457 Pseudomonascongelans Bacteria 200452 Pseudomonas Bacteria 53409 coronafaciensPseudomonas corrugata Bacteria 47879 Pseudomonas costantinii Bacteria168469 Pseudomonas Bacteria 157783 cremoricolorata Pseudomonas cremorisBacteria 2724178 Pseudomonas crudilactis Bacteria 2697028 PseudomonasBacteria 543360 cuatrocienegasensis Pseudomonas cyclaminis Bacteria2781239 Pseudomonas daroniae Bacteria 2487519 Pseudomonas Bacteria882211 deceptionensis Pseudomonas defluvii Bacteria 1876757 Pseudomonasdelhiensis Bacteria 366289 Pseudomonas denitrificans Bacteria 43306Pseudomonas Bacteria diazotrophicus Pseudomonas Bacteria 135830diterpeniphila Pseudomonas donghuensis Bacteria 1163398 Pseudomonasdryadis Bacteria 2487520 Pseudomonas duriflava Bacteria 459528Pseudomonas edaphica Bacteria 2006980 Pseudomonas ekonensis Bacteria2842353 Pseudomonas elodea Bacteria 179878 Pseudomonas endophyticaBacteria 1563157 Pseudomonas entomophila Bacteria 312306 Pseudomonaseucalypticola Bacteria 2599595 Pseudomonas excibis Bacteria PseudomonasBacteria 359110 extremaustralis Pseudomonas Bacteria 169669extremorientalis Pseudomonas fakonensis Bacteria 2842355 Pseudomonasfarris Bacteria 2841207 Pseudomonas farsensis Bacteria 2745492Pseudomonas ficuserectae Bacteria 53410 Pseudomonas fildesensis Bacteria1674920 Pseudomonas flavescens Bacteria 29435 Pseudomonas flexibilisBacteria 706570 Pseudomonas floridensis Bacteria 1958950 Pseudomonasfluorescens Bacteria 294 Pseudomonas fluvialis Bacteria 1793966Pseudomonas foliumensis Bacteria 2762593 Pseudomonas fragi Bacteria 296Pseudomonas Bacteria 104087 frederiksbergensis Pseudomonas fulgidaBacteria 200453 Pseudomonas fulva Bacteria 47880 Pseudomonas furukawaiiBacteria 1149133 Pseudomonas fuscovaginae Bacteria 50340 Pseudomonasgelidicola Bacteria 1653853 Pseudomonas gessardii Bacteria 78544Pseudomonas gingeri Bacteria 117681 Pseudomonas glareae Bacteria 1577705Pseudomonas glycinae Bacteria 1785145 Pseudomonas gozinkensis Bacteria2774461 Pseudomonas graminis Bacteria 158627 Pseudomonas granadensisBacteria 1421430 Pseudomonas Bacteria 1628277 gregormendelii Pseudomonasgrimontii Bacteria 129847 Pseudomonas Bacteria 1245526 guangdongensisPseudomonas Bacteria 1288410 guariconensis Pseudomonas guezenneiBacteria 310348 Pseudomonas guguanensis Bacteria 1198456 Pseudomonasguineae Bacteria 425504 Pseudomonas guryensis Bacteria 2759165Pseudomonas haemolytica Bacteria 2600065 Pseudomonas Bacteria 53411halodenitrificans Pseudomonas halodurans Bacteria 28258 PseudomonasBacteria halosaccharolytica Pseudomonas Bacteria halosensibilisPseudomonas hamedanensis Bacteria 2745504 Pseudomonas helianthi Bacteria251654 Pseudomonas helleri Bacteria 1608996 Pseudomonas Bacteria 1471381helmanticensis Pseudomonas huaxiensis Bacteria 2213017 Pseudomonashunanensis Bacteria 1247546 Pseudomonas hutmensis Bacteria 2707027Pseudomonas Bacteria 297 hydrogenothermophila Pseudomonas Bacteria 39439hydrogenovora Pseudomonas hydrolytica Bacteria 2493633 Pseudomonasindica Bacteria 137658 Pseudomonas indoloxydans Bacteria 404407Pseudomonas inefficax Bacteria 2078786 Pseudomonas iranensis Bacteria2745503 Pseudomonas iridis Bacteria 2710587 Pseudomonas izuensisBacteria 2684212 Pseudomonas japonica Bacteria 256466 Pseudomonasjessenii Bacteria 77298 Pseudomonas jinanensis Bacteria Pseudomonasjinjuensis Bacteria 198616 Pseudomonas juntendi Bacteria 2666183Pseudomonas Bacteria 2293832 kairouanensis Pseudomonas karstica Bacteria1055468 Pseudomonas Bacteria 2745482 kermanshahensis Streptococcusuberis Bacteria 1349 Besnoitia besnoiti Chromista 94643 Bonamia exitiosaChromista 362532 Bonamia ostreae Chromista 126728 Amniculicolalongissima Fungus 2566060 Arthroderma amazonicum Fungus 1592210Aschersonia hypocreoidea Fungus 370936 Aspergillago clavatoflava Fungus41064 Aspergillus acidohumus Fungus 1904037 Aspergillus acidus Fungus1069201 Aspergillus aculeatinus Fungus 487661 Aspergillus aculeatusFungus 5053 Aspergillus aeneus Fungus 41754 Aspergillus affinis Fungus1070780 Aspergillus alabamensis Fungus 657433 Aspergillus alliaceusFungus 209559 Aspergillus amazonicus Fungus 710228 Aspergillus ambiguusFungus 176160 Aspergillus amoenus Fungus 1220191 Aspergillus Fungus296546 amyloliquefaciens Aspergillus amylovorus Fungus 176161Aspergillus angustatus Fungus 2783700 Aspergillus anomalus Fungus 454240Aspergillus anthodesmis Fungus 37233 Aspergillus apicalis Fungus 478867Aspergillus Fungus 1140386 appendiculatus Aspergillus arachidicolaFungus 656916 Aspergillus ardalensis Fungus 1458899 Aspergillus arviiFungus 368784 Aspergillus Fungus 1695225 askiburgiensis Aspergillusasperescens Fungus 176163 Aspergillus assulatus Fungus 1245746Aspergillus astellatus Fungus 1810904 Aspergillus Fungus 41725aurantiobrunneus Aspergillus Fungus 2663348 aurantiopurpureusAspergillus aureolatus Fungus 41755 Aspergillus aureoterreus Fungus41288 Aspergillus aureus Fungus 309747 Aspergillus auricomus Fungus138274 Aspergillus austr aliensis Fungus 1250384 Aspergillusaustroafricanus Fungus 1220192 Aspergillus avenaceus Fungus 36643Aspergillus awamori Fungus 105351 Aspergillus baarnensis Fungus 2070749Aspergillus baeticus Fungus 1194636 Aspergillus bahamensis Fungus 522521Aspergillus bertholletiae Fungus 1226010 Aspergillus biplanus Fungus176164 Aspergillus bisporus Fungus 41753 Aspergillus bombycis Fungus109264 Aspergillus botswanensis Fungus 1810893 Candida albicans Fungus5476 Candida glabrata Fungus 5478 Candida krusei Fungus 4909 Candidaparapsilosis Fungus 5480 Candida tropicalis Fungus 5482 Cryptococcusgattii Fungus 37769 Cryptococcus neoformans Fungus 5207 EpidermophytonFungus 34391 floccosum Epidermophyton Fungus 74042 stockdaleae Fusariumacaciae Fungus Fusarium acaciae-mearnsii Fungus 282272 Fusarium acicolaFungus Fusarium acremoniopsis Fungus Fusarium acridiorum Fungus Fusariumacutatum Fungus 78861 Fusarium aderholdii Fungus Fusarium adesmiaeFungus Fusarium aduncisporum Fungus Fusarium aecidii- Fungustussilaginis Fusarium aeruginosum Fungus Fusarium aethiopicum Fungus569394 Fusarium affine Fungus Fusarium agaricorum Fungus Fusariumailanthinum Fungus Fusarium alabamense Fungus Fusarium albedinis FungusFusarium albertii Fungus Fusarium Fungus albidoviolaceum Fusariumalbiziae Fungus Fusarium albocarneum Fungus Fusarium album FungusFusarium aleurinum Fungus Fusarium aleyrodis Fungus Fusariumalkanophilum Fungus Fusarium allescheri Fungus Fusarium allescherianumFungus Fusarium allii-sativi Fungus Trichophyton simii Fungus 63406Trichophyton Fungus 69891 soudanense Trichophyton tonsurans Fungus 34387Trichophyton verrucosum Fungus 63417 Trichophyton violaceum Fungus 34388Ochroma pyramidale Plant 66662 Babesia bigemina Protozoa 5866 Babesiabovis Protozoa 5865 Babesia divergens Protozoa 32595 Babesia jakimoviProtozoa Babesia major Protozoa 127461 Babesia occultans Protozoa 536930Babesia ovata Protozoa 189622 Cryptosporidium parvum Protozoa 5807Eimeria acervulina Protozoa 5801 Eimeria brunetti Protozoa 51314 Eimeriamaxima Protozoa 5804 Eimeria meleagridis Protozoa 1431345 Eimerianecatrix Protozoa 51315 Eimeria tenella Protozoa 5802 Entamoeba Protozoa5759 histolytica Giardia duodenalis Protozoa 5741 Giardia lambiaProtozoa Histomonas meleagridis Protozoa 135588 Ichthyobodo necatorProtozoa 155203 Ichthyophthirius Protozoa 5932 multifiliis Isosporaburrowsi Protozoa Isospora canis Protozoa 1662860 Isospora felisProtozoa 482539 Isospora neorivolta Protozoa Isospora ohioensis Protozoa279926 Leishmania braziliensis Protozoa 5660 Leishmania chagasi Protozoa44271 Leishmania infantum Protozoa 5671 Marteilia refringens Protozoa107386 Mikrocytos mackini Protozoa 195010 Perkinsus marinus Protozoa31276 Perkinsus olensi Protozoa Sarcocystis cruzi Protozoa 5817Sarcocystis hirsuta Protozoa 61649 Sarcocystis hominis Protozoa 61650Theileria annulata Protozoa 5874 Theileria buffei Protozoa Theilerialestoquardi Protozoa 77054 Theileria luwenshuni Protozoa 540482Theileria mutans Protozoa 27991 Theileria orientalis Protozoa 68886Theileria parva Protozoa 5875 Theileria sergenti Protozoa 5877 Theileriauilenbergi Protozoa 507731 Toxoplasma gondii Protozoa 5811 Trichomonasfetus Protozoa Trichomonas gallinae Protozoa 56777 Trichomonas stableriProtozoa 1440121 Trypanosoma brucei Protozoa 5691 Trypanosoma congolenseProtozoa 5692 Trypanosoma cruzi Protozoa 5693 Abras virus Virus 2303487Absettarov virus Virus Abu Hammad virus Virus 248058 Abu Mina virusVirus 248059 Acado virus Virus Acara virus Virus 2748201 Achiote virusVirus 2036702 Adana virus Virus 1611877 Adelaide River virus Virus 31612Adria virus Virus Aedes aegypti densovirus Virus 186156 Aedes albopictusVirus 35338 densovirus Aedes flavivirus Virus 390845 Aedes galloisiflavivirus Virus 1046551 Aedes pseudoscutellaris Virus densovirus Aedespseudoscutellaris Virus 341721 reovirus Aedes vexans Virus 7163 Africanhorse sickness Virus 40050 virus African swine fever virus Virus 10497Aguacate virus Virus 1006583 Aino virus Virus 11582 Akabane virus Virus70566 Alajuela virus Virus 1552846 Alcelaphine Virus 35252gammaherpesvirus 1 Alenquer virus Virus 629726 Aleutian Mink DiseaseVirus Alfuy virus Virus 44017 Alkhumra hemorrhagic Virus 172148 fevervirus Allpahuayo Virus 144752 mammarenavirus Almeirim virus VirusAlmendravirus arboretum Virus 1972683 Almendravirus cootbay Virus1972685 Almpiwar virus Virus 318843 Alocasia macrorrhizos Virus 4456Altamira virus Virus Amapari virus Virus Ambe virus Virus 1926500 Amgavirus Virus 1511732 Amur/Soochong virus Virus Anadyr virus Virus 1642852Anajatuba virus Virus 379964 Ananindeua virus Virus 1927813 Andasibevirus Virus Andes orthohantavirus Virus 1980456 Anhanga virus Virus904722 Anhembi virus Virus 273355 Anopheles A virus Virus 35307Anopheles B virus Virus 35308 Anopheles flavivirus Virus 2053814Anopheles gambiae Virus 487311 densovirus Antequera virus Virus 2748239Apoi virus Virus 64280 Araguari virus Virus 352236 Aransas Bay virusVirus 1428582 Araraquara virus Virus 139032 Bluetongue virus Virus 40051Bobaya virus Virus 2818228 Bobia virus Virus Boraceia virus Virus Bornadisease virus Virus 12455 Botambi virus Virus Boteke virus Virus 864698Bouboui virus Virus 64295 Bourbon virus Virus 1618189 Bovine ephemeralfever Virus 11303 virus Bovine Herpes Virus 1 Virus Bovine leukemiavirus Virus 11901 Bovine orthopneumovirus Virus 11246 Bovine viral Virus11099 diarrhea virus 1 Bowe virus Virus 1400425 Bozo virus Virus 273349Cumuto virus Virus 1457166 Cupixi mammarenavirus Virus 208899Curionopolis virus Virus 490110 Cyprinid herpesvirus 3 Virus 180230Czech Aedes vexans Virus flavivirus virus D’Aguilar virus Virus Dabakalavirus Virus Dabieshan virus Virus 1167310 Dak Nong virus Virus 1238455Dakar bat virus Virus 64282 Dandenong virus Virus 483046 Dashli virusVirus 1764087 Deer tick virus Virus 58535 Dengue virus Virus 12637Dengue virus 1 virus Virus Cumuto virus Virus 1457166 Cupiximammarenavirus Virus 208899 Curionopolis virus Virus 490110 LymphocyticVirus 11623 choriomeningitis mammarenavirus Lyssavirus aravan Virus211977 Lyssavirus australis Virus 90961 Lyssavirus lagos Virus 38766Lyssavirus spp. Virus 11286 Lyssavirus bokeloh Virus 1072176 Lyssaviruscaucasicus Virus 249584 Lyssavirus duvenhage Virus 38767 Lyssavirusirkut Virus 249583 Lyssavirus khujand Virus 237716 Lyssavirus mokolaVirus 12538 Lyssavirus rabies Virus 11292 Lyssavirus shimoni Virus746543 Marisma mosquito virus Virus 1105173 Marituba virus Virus 292278Marondera virus Virus 108092 Marrakai virus Virus 108088 Massila virusVirus Matariya virus Virus 1272948 Matruh virus Virus 1678229 Matucarevirus Virus 908873 Mayaro virus Virus 59301 Mboke virus Virus 273342Mburo virus Virus 2035534 Meaban virus Virus 35279 Medjerda Valley virusVirus 1775957 Melao virus Virus 35515 Meno virus Virus Mercadeo virusVirus 1708574 Semliki Forest virus Virus 11033 Sena Madureira virusVirus 1272957 Seoul virus Virus 1980490 Sepik virus Virus 44026 Serra DoNavio virus Virus 45768 Serra Norte virus Virus 1000649 Severe feverwith Virus 1003835 thrombocytopenia syndrome virus Shamonda virus Virus159150 Shark River virus Virus 2303490 Shiant Island virus Virus Shokwevirus Virus 273359 Shuni virus Virus 159148 Silverwater virus Virus1564099 Simbu orthobunyavirus Virus 35306 Sin Nombre virus Virus 1980491Sindbis virus Virus 11034 Sixgun City virus Virus Skinner Tank virusVirus 481886 Snowshoe hare virus Virus 11580 Sokoluk virus Virus 64317Soldado virus Virus 426791 Solwezi virus Virus Somone virus VirusSororoca virus Virus 273354 Souris virus Virus 2010246 South Bay virusVirus 1526514 South River virus Virus 45769 Spanish Culex flavivirusVirus virus Spanish Ochlerotatus Virus flavivirus virus Spondweni virusVirus 64318 Sprivirus cyprinus Virus 696863 Sripur virus Virus 1620897St. Abbs Head virus Virus St. Croix River virus Virus St. Louisencephalitis Virus 11080 virus Stanfield virus Virus Stratford virusVirus 44027

TABLE 6 Exemplary list of viruses NCBI Taxonomy Name ID Aalivirus A2169685 Aarhusvirus 2732762 dagda Aarhusvirus 2732763 katbat Aarhusvirus2732764 luksen Aarhusvirus 2732765 mysterion Abaca bunchy 438782 topvirus Abatino 2734574 macacapox virus Abbeymikolon- 2734213 virusabbeymikolon Abouovirus 1984774 abouo Abouovirus 1984775 davies Abutilon1926117 golden mosaic virus Abutilon 932071 mosaic Bolivia virusAbutilon 1046572 mosaic Brazil virus Abutilon 10815 mosaic virusAbutilon 169102 yellows virus Acadevirus 2733576 PM116 Acadevirus2733577 Pm5460 Acadevirus 2733574 PM85 Acadevirus 2733575 PM93Acadianvirus 1982901 acadian Acadianvirus 1982902 baee Acadianvirus1982903 reprobate Acanthamoeba 212035 polyphaga mimivirus Acanthocystis322019 turfacea chlorella virus 1 Acara 2170053 orthobunyavirus Achimota2560259 pararubulavirus 1 Achimota 2560260 pararubulavirus 2Achromobacter 2169962 virus Axp3 Acidianus 437444 bottle-shaped virusAcidianus 300186 filamentous virus 2 Acidianus 346881 filamentous virus3 Acidianus 346882 filamentous virus 6 Acidianus 346883 filamentousvirus 7 Acidianus 346884 filamentous virus 8 Acidianus 512792filamentous virus 9 Acidianus 309181 rod-shaped virus 1 Acidianus 693629spindle- shaped virus 1 Acidianus 315953 two-tailed virus Acinetobacter279006 virus 133 Acintetobacter virus B2 Acintetobacter virus B5Acionnavirus 2734078 monteraybay Acipenserid 2871198 herpesvirus 2Aconitum 101764 latent virus Acrobasis zelleri entomopoxvirus Actinidiaseed 2560282 borne latent virus Actinidia 2024724 virus 1 Actinidia1112769 virus A Actinidia 1112770 virus B Actinidia 1331744 virus XAcute bee 92444 paralysis virus Adana 2734433 phlebovirus Adeno- 1511891associated dependoparvo virus A Adeno- 1511892 associated dependoparvovirus B Adoxophyes 1993630 honmai entomopoxvirus Adoxophyes 224399honmai nucleopolyhedro- virus Adoxophyes 170617 orana granulovirus Aedesaegypti entomopoxvirus Aedes aegypti Mosqcopia virus Aedes 341721pseudoscutellaris reovirus Aegirvirus 2733888 SCBP42 Aeonium 1962503ringspot virus Aeromonas virus 43 Aeropyrum 1157339 coil-shaped virusAeropyrum 700542 pernix bacilliform virus 1 Aeropyrum 1032474 pernixovoid virus 1 Aerosvirus 2733365 AS7 Aerosvirus 2733364 av25AhydR2PPAerosvirus 2733366 ZPAH7 Affertcholeram- 141904 virus CTXphi African2560285 cassava mosaic Burkina Faso virus African 10817 cassava mosaicvirus African 2056161 eggplant mosaic virus African horse 40050 sicknessvirus African oil 185218 palm ringspot virus African swine 10497 fevervirus Agaricus 2734345 bisporus alphaendorna- virus 1 Agaricus bisporusvirus 4 Agatevirus 1910935 agate Agatevirus 1910936 bobb Agatevirus1910937 Bp8pC Ageratum 1260769 enation alphasatellite Ageratum 188333enation virus Ageratum 1386090 latent virus Ageratum leaf 912035 curlBuea betasatellite Ageratum leaf 635076 curl Cameroon betasatelliteAgeratum leaf 2182585 curl Sichuan virus Ageratum leaf 333293 curl virusAgeratum 169687 yellow leaf curl betasatellite Ageratum 187850 yellowvein alphasatellite Ageratum 185750 yellow vein betasatellite Ageratum1454227 yellow vein China alphasatellite Ageratum 437063 yellow veinHualian virus Ageratum 1407058 yellow vein India alphasatellite Ageratum2010316 yellow vein India betasatellite Ageratum 915293 yellow veinSingapore alphasatellite Ageratum 2010317 yellow vein Sri Lankabetasatellite Ageratum 222079 yellow vein Sri Lanka virus Ageratum 44560yellow vein virus Aghbyvirus 2733367 ISAO8 Aglaonema 1512278 bacilliformvirus Agricanvirus 1984777 deimos Agricanvirus 2560433 desertfoxAgricanvirus 1984778 Ea3570 Agricanvirus 1984779 ray Agricanvirus1984780 simmy50 Agricanvirus 1984781 specialG Agropyron 41763 mosaicvirus Agrotis 208013 ipsilon multiple nucleopolyhed rovirus Agrotis10464 segetum granulovirus Agrotis 1962501 segetum nucleopolyhed rovirusA Agrotis 1580580 segetum nucleopolyhed rovirus B Agtrevirus 1987994 AG3Agtrevirus 2169690 SKML39 Aguacate 2734434 phlebovirus Ahlum waterbornevirus Ahphunavirus 2733368 Ahp1 Ahphunavirus 2733369 CF7 Ahtivirus2734079 sagseatwo Aichivirus A 72149 Aichivirus B 194965 Aichivirus C1298633 Aichivirus D 1897731 Aichivirus E 1986958 Aichivirus F 1986959Ailurivirus A 2560287 Aino 2560289 orthobunyavirus Air potato 2560290ampelovirus 1 Akabane 1933178 orthobunyavirus Akhmeta virus 2200830Alajuela 1933181 orthobunyavirus Alasvirus 2501934 muscae Alcelaphine35252 gammaherpes virus 1 Alcelaphine 138184 gammaherpes virus 2 Alcube2734435 phlebovirus Alcyoneusvirus 2560541 K641 Alcyoneusvirus 2560545RaK2 Alefpapilloma 2169692 virus 1 Alenquer 2734436 phlebovirusAlexandravirus 2734080 AD1 Alexandravirus 2734081 alexandra Alfalfabetanucleorha bdovirus Alfalfa cryptic virus 1 Alfalfa 1770265enamovirus 1 Alfalfa leaf 1306546 curl virus Alfalfa mosaic 12321 virusAlfalfa virus S 1985968 Algerian 515575 watermelon mosaic virusAllamanda 452758 leaf curl virus Allamanda 1317107 leaf mottledistortion virus Alligatorweed stunting virus Allium cepa 2058778amalgavirus 1 Allium cepa 2058779 amalgavirus 2 Allium virus 317027 XAllpahuayo 144752 mammarenavius Almendravirus 1972686 almendrasAlmendravirus 1972683 arboretum Almendravirus 1972684 balsaAlmendravirus 1972687 chico Almendravirus 1972685 cootbay Almendravirus2734366 menghai Bat associated 1987731 cyclovirus 6 Bat associated1987732 cyclovirus 7 Bat associated 1987733 cyclovirus 8 Bat associated1987734 cyclovirus 9 Bat 1913643 coronavirus CDPHE15 Bat 1244203coronavirus HKU10 Bat Hp- 2501961 betacoronavirus Zhejiang2013 Bat1146877 mastadenovirus A Bat 1146874 mastadenovirus B Bat 2015370mastadenovirus C Bat 2015372 mastadenovirus D Bat 2015374 mastadenovirusE Bat 2015375 mastadenovirus F Bat 2015376 mastadenovirus G Batmastadenovirus H Bat mastadenovirus I Bat mastadenovirus J Batai 2560341orthobunyavirus Batama 1933177 orthobunyavirus Batfish 2560342actinovirus Bavaria virus 2560343 Baxtervirus 2169730 baxterfoxBaxtervirus 2169731 yeezy Baylorvirus 2734055 bv1127AP1 Baylorvirus376820 PHL101 Bayou 1980459 orthohantavirus Bcepfunavirus 417280 bcepF1Bcepmuvirus 264729 bcepMu Bcepmuvirus 431894 E255 Bdellomicrovirus1986027 MH2K Bdellovibrio virus MAC1 Beak and 77856 feather diseasevirus Bean calico 31602 mosaic virus Bean chlorosis 1227354 virus Beancommon 43240 mosaic necrosis virus Bean common 12196 mosaic virus Beandwarf 10838 mosaic virus Bean golden 10839 mosaic virus Bean golden220340 yellow mosaic virus Bean leaf 2004460 crumple virus Bean leafroll12041 virus Bean mild mosaic virus Bean necrotic 2560344 mosaicorthotospovirus Bean pod 12260 mottle virus Bean rugose 128790 mosaicvirus Bean white 2169732 chlorosis mosaic virus Bean yellow 267970disorder virus Bean yellow 714310 mosaic Mexico virus Bean yellow 12197mosaic virus Bear Canyon 192848 mammarenavirus Beauveria 1740646bassiana polymycovirus 1 Beauveria 1685109 bassiana victorivirus 1Bebaru virus 59305 Beecentumtre 10778 virus B103 Beet black 196375scorch virus Beet chlorosis 131082 virus Beet cryptic 509923 virus 1Beet cryptic 912029 virus 2 Beet cryptic 29257 virus 3 Beet curly top391228 Iran virus Beet curly top 10840 virus Beet mild 156690 yellowingvirus Beet mosaic 114921 virus Beet necrotic 31721 yellow vein virusBeet 72750 pseudoyellows virus Beet ringspot 191547 virus Beet soil-76343 borne mosaic virus Beet soil- 46436 borne virus Beet virus Q 71972Beet western 12042 yellows virus Beet yellow 35290 stunt virus Beetyellows 12161 virus Beetle mivirus Beetrevirus 2560656 B3 Beetrevirus2560663 JBD67 Beetrevirus 2560664 JD18 Beetrevirus 2560675 PM105 Beihaipicobirnavirus Beilong 2560345 jeilongvirus Bell pepper 354328alphaendorna- virus Bell pepper 368735 mottle virus Belladonna 12149mottle virus Bellamyvirus 2734095 bellamy Bellavista 2560346orthobunyavirus Bellflower 1720595 vein chlorosis virus Bellflower1982660 veinal mottle virus Beluga whale 694015 coronavirus SW1Bendigovirus 2560495 GMA6 Benedictvirus 1071502 cuco Benedictvirus1993876 tiger Benevides 2170054 orthobunyavirus Bequatrovirus 1984785avesobmore Bequatrovirus 1918005 B4 Bequatrovirus 1918006 bigberthaBequatrovirus 1918007 riley Bequatrovirus 1918008 spock Bequatrovirus1918009 troll Berhavirus 2509379 beihaiense Berhavirus 2509380 radialisBerhavirus 2509381 sipunculi Berisnavirus 1 2734518 Cacao yellow 12150mosaic virus Cacao yellow 2169726 vein banding virus Cache Valley2560364 orthobunyavirus Cachoeira 2560365 Porteira orthobunyavirusCacipacore 64305 virus Cactus mild 229030 mottle virus Cactus virus 2Cactus virus X 112227 Cadicivirus A 1330068 Cadicivirus B 2560366Caenorhabditis elegans Cer1 virus Caenorhabditis elegans Cer13 virusCaeruleovirus 1985175 Bc431 Caeruleovirus 1985176 Bcp1 Caeruleovirus1985177 BCP82 Caeruleovirus 1985178 BM15 Caeruleovirus 1985179 deepblueCaeruleovirus 1985180 JBP901 Cafeteria 1513235 roenbergensis virusCafeteriavirus- 1932923 dependent mavirus Caimito 2734421 pacuvirusCajanus cajan Panzee virus Caladenia 1198147 virus A Calanthe mild 73840mosaic virus Cali 2169993 mammarenavirus Calibrachoa 204928 mottle virusCalifornia 1933264 encephalitis orthobunyavirus California 2170175reptarenavirus Caligid hexartovirus Caligrhavirus 2560367 caligusCaligrhavirus 2560551 lepeophtheirus Caligrhavirus 2560736 salmonlouseCalla lily 2560368 chlorotic spot orthotospovirus Calla lily 243560latent virus Callistephus 1886606 mottle virus Callitrichine 106331gammaherpes virus 3 Calopogonium yellow vein virus Camel 2169876associated drosmacovirus 1 Camel 2169877 associated drosmacovirus 2Camel 2170105 associated porprismaco- virus 1 Camel 2170106 associatedporprismaco- virus 2 Camel 2170107 associated porprismaco- virus 3 Camel2170108 associated porprismaco- virus 4 Camelpox 28873 virus Campana2734442 phlebovirus Campoletis aprilis ichnovirus Campoletis flavicinctaichnovirus Camptochiron omus tentans entomopoxvirus Campylobacter1006972 virus IBB35 Camvirus 1982882 amela Camvirus 1982883 CAM Canary142661 circovirus Canarypox 44088 virus Candida albicans Tca2 virusCandida albicans Tca5 virus Candiru 1933182 phlebovirus Canid 170325alphaherpesvirus 1 Canine 1985425 associated gemygorvirus 1 Canine1194757 circovirus Canine 10537 mastadenovirus A Canine 11232morbillivirus Canna yellow 2560371 mottle associated virus Canna yellow419782 mottle virus Canna yellow 433462 streak virus Cannabis 1115692cryptic virus Cano 1980463 Delgadito orthohantavirus Canoevirus 2734056canoe Cao Bang 1980464 orthohantavirus Caper latent 1031708 virus Capim1933265 orthobunyavirus Capistrivirus 2011077 KSF1 Capraria 2049955yellow spot virus Caprine 39944 alphaherpesvirus 1 Caprine 11660arthritis encephalitis virus Caprine 135102 gammaherpes virus 2 Caprine2560372 respirovirus 3 Capsicum 2560373 chlorosis orthotospovirusCapsicum 2734586 India alphasatellite Captovirus 235266 AFV1 Capuchin2163996 monkey hepatitis B virus Caraparu 1933290 orthobunyavirusCarbovirus 2136037 queenslandense Dyonupapillo 1513250 mavirus 1Dyoomega- 1918731 papillomavirus 1 Dyoomikron- 1513251 papillomavirus 1Dyophipapilloma- 1920493 virus 1 Dyopipapilloma- 1513252 virus 1Dyopsipapilloma- 1920498 virus 1 Dyorhopapilloma- 1513253 virus 1Dyosigmapapilloma- 1513254 virus 1 Dyotau- 1932910 papillomavirus 1Dyotheta- 1235662 papillomavirus 1 Dyoupsilon- 1932912 papillomavirus 1Dyoxipapilloma- 1513255 virus 1 Dyoxipapilloma- 2169881 virus 2 Dyozeta-1177766 papillomavirus 1 Eapunavirus 2733615 Eap1 East African 223262cassava mosaic Cameroon virus East African 393599 cassava mosaic Kenyavirus East African 223264 cassava mosaic Malawi virus East African 62079cassava mosaic virus East African 223275 cassava mosaic Zanzibar virusEast Asian 2734556 Passiflora distortion virus East Asian 341167Passiflora virus Eastern 2170195 chimpanzee simian foamy virus Easternequine 11021 encephalitis virus Eastern 2734571 kangaroopox virusEastlansingvirus 2734004 Sf12 Echarate 2734447 phlebovirus Echinochloa42630 hoja blanca tenuivirus Echinochloa ragged stunt virus Ecliptayellow 2030126 vein alphasatellite Eclipta yellow 875324 vein virusEclunavirus 2560414 EcL1 Ectocarpus 2083183 fasciculatus virus aEctocarpus 37665 siliculosus virus 1 Ectocarpus siliculosus virus aEctromelia 12643 virus Ectropis 59376 obliqua nucleopolyhedro- virusEctropis 1225732 obliqua virus Edenvirus 2734230 eden Edge Hill 64296virus Efquatrovirus 2560415 AL2 Efquatrovirus 2560416 AL3 Efquatrovirus2560417 AUEF3 Efquatrovirus 2560424 EcZZ2 Efquatrovirus 2560420 EF3Efquatrovirus 2560421 EF4 Efquatrovirus 2560425 EfaCPT1 Efquatrovirus2560426 IME196 Efquatrovirus 2560427 LY0322 Efquatrovirus 2560428 PMBT2Efquatrovirus 2560429 SANTOR1 Efquatrovirus 2560430 SHEF2 Efquatrovirus2560431 SHEF4 Efquatrovirus 2560432 SHEF5 Eganvirus EtG 2734059Eganvirus 29252 ev186 Enterovirus A 138948 Enterovirus B 138949Enterovirus C 138950 Enterovirus D 138951 Enterovirus E 12064Enterovirus F 1330520 Enterovirus G 106966 Enterovirus H 310907Enterovirus I 2040663 Enterovirus J 1330521 Enterovirus K 2169884Enterovirus L 2169885 Entnonaginta- 2734061 virus ENT90 Entoleuca2734428 entovirus Enytus montanus ichnovirus Ephemerovirus 1972589adelaide Ephemerovirus 1972594 berrimah Ephemerovirus 1972593 febrisEphemerovirus 1972595 kimberley Ephemerovirus 1972596 koolpinyahEphemerovirus 1972587 kotonkan Ephemerovirus 1972592 obodhiangEphemerovirus 1972597 yata Epichloe 382962 festucae virus 1 Epinotia166056 aporema granulovirus Epiphyas 70600 postvittana nucleopolyhedrovirus Epirus cherry 544686 virus Epizootic 100217 haematopoieticnecrosis virus Epizootic 40054 hemorrhagic disease virus Eponavirus2734105 epona Epseptimavirus 1982565 118970sal2 Epseptimavirus 491003EPS7 Epseptimavirus 2732021 ev123 Epseptimavirus 2732022 ev329Epseptimavirus 2732023 LVR16A Epseptimavirus 2732019 mar003J3Epseptimavirus 2732024 S113 Epseptimavirus 2732025 S114 Epseptimavirus2732026 S116 Epseptimavirus 2732027 S124 Epseptimavirus 2732028 S126Epseptimavirus 2732029 S132 Epseptimavirus 2732030 S133 Epseptimavirus2732031 S147 Epseptimavirus 2732020 saus132 Epseptimavirus 2732032seafire Epseptimavirus 2732033 SH9 Epseptimavirus 2732034 STG2Epseptimavirus 1540099 stitch Epseptimavirus 2732035 Sw2Epsilonarterivirus 2501964 hemcep Epsilonarterivirus 2501965 safriverEpsilonarterivirus 2501966 zamalb Epsilonpapilloma- 40537 virus 1Epsilonpapilloma- 2169886 virus 2 Epsilonpolyoma- 1891754 virus bovisEptesipox 1329402 virus Equid 10326 alphaherpesvirus 1 Equid 80341alphaherpesvirus 3 Equid 10331 alphaherpesvirus 4 Equid 39637alphaherpesvirus 8 Equid 55744 alphaherpesvirus 9 Equid 12657gammaherpes virus 2 Equid 10371 gammaherpes virus 5 Equid 291612gammaherpes virus 7 Equine 1985379 associated gemycircular- virus 1Equine 201490 encephalosis virus Equine foamy 109270 virus Equine 11665infectious anemia virus Equine 129954 mastadenovirus A Equine 129955mastadenovirus B Equine 2723956 picobirnavirus Equine rhinitis 47000 Avirus Equine 329862 torovirus Eracentumvirus 1985737 era103Eracentumvirus 2733579 S2 Eragrostis 638358 curvula streak virusEragrostis 1030595 minor streak virus Eragrostis 496807 streak virusErbovirus A 312185 Erectites 390443 yellow mosaic virus Eriborusterebrans ichnovirus Erinnyis ello 307444 granulovirus Eriocheir 273810sinensis reovirus Ermolevavirus 2733903 PGT2 Ermolevavirus 2733904 PhiKTErskinevirus 2169882 asesino Erskinevirus 2169883 EaH2 Erysimum 12152latent virus Feline 1987742 associated cyclovirus 1 Feline 11978calicivirus Feline foamy 53182 virus Feline 11673 immunodeficiency virusFeline 11768 leukemia virus Feline 1170234 morbillivirus Felipivirus AFelixounavirus 2560439 Alf5 Felixounavirus 1965378 AYO145AFelixounavirus 2560723 BPS15Q2 Felsduovirus 2734062 4LV2017 Felsduovirus194701 Fels2 Felsduovirus 2734063 RE2010 Felsduovirus 2734062 4LV2017Felsduovirus 194701 Fels2 Fernvirus 1921560 shelly Fernvirus 1921561sitara Festuca leaf streak cytorhabdovirus Fibralongavirus 2734233fv2638A Fibralongavirus 2734234 QT1 Fibrovirus fs1 70203 Fibrovirus1977140 VGJ Ficleduovirus 2560473 FCL2 Ficleduovirus 2560474 FCV1 Figbadnavirus 1 1034096 Fig cryptic 882768 virus Figulus sublaevisentomopoxvirus Figwort 10649 mosaic virus Fiji disease 77698 virus Finch400122 circovirus Finkel-Biskis- 353765 Jinkins murine sarcoma virusFinnlakevirus 2734591 FLiP Fionnbharthvirus 2955891 fionnbharthFipivirus A Fipvunavirus 2560476 Fpv4 Firehammervirus 1190451 CP21Firehammervirus 722417 CP220 Firehammervirus 722418 CPt10 Fischettivirus230871 C1 Fishburnevirus 1983737 brusacoram Flamingopox 503979 virusFlammulina 568090 velutipes browning virus Flaumdravirus 2560665 KIL2Flaumdravirus 2560666 KIL4 Fletchervirus 1980966 CP30A Gaiavirus gaia1982148 Gaillardia 1468172 latent virus Gairo 1535802 mammarenavirusGajwadongvirus 2733916 ECBP5 Gajwadongvirus 2733917 PP99 Galaxyvirus2560298 abidatro Galaxyvirus 2560303 galaxy Galinsoga 60714 mosaic virusGallid 10386 alphaherpesvirus 1 Gamaleyavirus 1920761 Sb1 Gambievirus2501933 bolahunense Gamboa 1933270 orthobunyavirus Gammaarterivirus2499678 lacdeh Gammanucleo 2748968 rhabdovirus maydis Gammapapilloma-333926 virus 1 Gammapapilloma- 1175852 virus 10 Gammapapilloma- 1513256virus 11 Gayfeather 578305 mild mottle virus Gecko 2560481 reptillovirusGelderlandvirus 2560727 melville Gelderlandvirus 1913658 s16Gelderlandvirus 1913657 stml198 Gelderlandvirus 2560734 stp4a Gentian182452 mosaic virus Gentian ovary 1920772 ringspot virus Geotrupessylvaticus entomopoxvirus Gequatrovirus 1986034 G4 Gequatrovirus 1910968ID52 Gequatrovirus 1910969 talmos Gerygone 1985381 associatedgemycircular- virus 1 Gerygone 1985382 associated gemycircular- virus 2Harrisina 115813 brillians granulovirus Harrisonvirus 1982221 harrisonHarvey 11807 murine sarcoma virus Hautre virus 1982895 hau3 Havel River254711 virus Hawkeyevirus 2169910 hawkeye Hazara 1980522 orthonairovirusHeartland 2747342 banda virus Hebius tobanivirus 1 Hedgehog 1965093coronavirus 1 Hedwigvirus 2560502 hedwig Hedyotis 1428190 uncinellayellow mosaic virus Hedyotis 1428189 yellow mosaic betasatelliteHeilongjiangvirus 2734110 Lb Helenium 12171 virus S Helianthus 2184469annuus alphaendornavirus Helicobasidium 675833 mompa alphaendorna- virus1 Helicobasidium 344866 mompa partitivirus V70 Helicobasidium 196690mompa totivirus 1-17 Helicoverpa 489830 armigera granulovirusHelicoverpa 51313 armigera nucleopolyhedro- virus Helicoverpa 37206armigera stunt virus Heliothis 10290 armigera entomopoxvirus Heliothis113366 virescens ascovirus 3a Heliothis zea 29250 nudivirus Helleborus592207 mosaic virus Helleborus net 592206 necrosis virus Helminthos-2560520 porium victoriae virus 145S Helminthos- 45237 porium victoriaevirus 190S Helsettvirus 2733626 fPS53 Helsettvirus 2733628 fPS54ocrHelsettvirus 2733627 fPS59 Helsettvirus 2733625 fPS9 Helsingorvirus1918193 Cba121 Helsingorvirus 1918194 Cba171 Jujube 2020956 mosaic-associated virus Jun 2560536 jeilongvirus Juncopox virus Jutiapa virus64299 Jwalphavirus 2169963 jwalpha Kabuto 2747382 mountain uukuvirusKadam virus 64310 Kadipiro virus 104580 Kaeng Khoi 1933275orthobunyavirus Kafavirus 2733923 SWcelC56 Kafunavirus 1982588 KF1Kagunavirus 2560464 golestan Kagunavirus 1911008 K1G Kagunavirus 1911010K1H Kagunavirus 1911007 Klind1 Kagunavirus 1911009 Klind2 Kagunavirus2734197 RP180 Merremia 77813 mosaic virus Mesta yellow 1705093 veinmosaic alphasatellite Mesta yellow 508748 vein mosaic Bahraich virusMetamorphoo 2734253 virus fireman Metamorphoo 2734254 virus metamorphooMetamorphoo 2734255 virus robsfeet Metrivirus 2560269 ME3 Mguuvirus2733593 JG068 Microbacterium virus MuffinTheCat [2] Microcystis 340435virus Ma- LMM01 Microhyla letovirus 1 Micromonas 338781 pusilia reovirusMicromonas 373996 pusilia virus SP1 Microplitis croceipes bracovirusMicrotus 2006148 arvalis polyomavirus 1 Mukerjeevirus 2734186 mv52B1Mulberry 1227557 badnavirus 1 Mulberry 1631303 mosaic dwarf associatedvirus Mulberry 1527441 mosaic leaf roll associated virus Mulberryringspot virus Mulberry vein banding associated orthotospovirus Muledeerpox 304399 virus Mume virus A 2137858 Mumps 2560602 orthorubulavirusMungbean 2010322 yellow mosaic betasatellite Mukerjeevirus 2734186mv52B1 Mulberry 1227557 badnavirus 1 Mulberry 1631303 mosaic dwarfassociated virus Mycobacterium 1993864 virus Tweety Mycobacterium1993860 virus Wee Mycobacterium 1993859 virus Wildcat Mycoreovirus 1311228 Mycoreovirus 2 404237 Mycoreovirus 3 311229 Mylasvirus 1914020persius Mynahpox 2169711 virus Myodes coronavirus 2JL14 Myodes 2006147glareolus polyomavirus 1 Myodes 2560609 jeilongvirus Myodes 2560610narmovirus Myohalovirus 1980944 phiH Noxifervirus 2560671 noxifer Ntayavirus 64292 Ntepes 2734464 phlebovirus Nuarterivirus guemel Nudaurelia85652 capensis beta virus Nudaurelia 12541 capensis omega virusNupapilloma- 334205 virus 1 Nyando 1933306 orthobunyavirus Nyavirus644609 midwayense Nyavirus 644610 nyamaniniense Nyavirus 1985708sierranevadaense Nyceiraevirus 2560506 nyceirae Nyctalus 2501928velutinus alphacoronavirus SC-2013 Nylanderia 1871153 fulva virus 1Nymphadoravirus 2170041 kita Nymphadoravirus 2560507 nymphadoraNymphadoravirus 2170042 zirinka Oat blue 56879 dwarf virus Oat chlorotic146762 stunt virus Oat dwarf 497863 virus Oat golden 45103 stripe virusOxbow 1980484 orthohantavirus Oxyplax 2083176 ochracea nucleopolyhedro-virus Paadamvirus 2733939 RHEph01 Pacific coast uukuvirus Pacui 2560617pacuvirus Paenibacillus virus Willow Pagavirus 2733940 S05C849 Pagevirus1921185 page Pagevirus 1921186 palmer Pagevirus 1921187 pascal Pagevirus1921188 pony Pagevirus 1921189 pookie Pagoda yellow 1505530 mosaicassociated virus Paguronivirus 1 2508237 Pahexavirus 1982252 ATCC29399BCPahexavirus 1982303 pirate Pahexavirus 1982304 procrass1 Pahexavirus1982305 SKKY Pahexavirus 1982306 solid Pahexavirus 1982307 stormbornPahexavirus 1982308 wizzo Pahsextavirus 2733975 pAh6C Pairvirus 2733941Lo5R7ANS Pakpunavirus 1921409 CAb02 Pahexavirus 1982303 piratePahexavirus 1982304 procrass1 Pahexavirus 1982305 SKKY Pea necrotic753670 yellow dwarf virus Pea seed- 12208 borne mosaic virus Pea stem199361 necrosis virus Pea streak 157777 virus Pea yellow 1436892 stuntvirus Peach 471498 chlorotic mottle virus Peach latent 12894 mosaicviroid Peach 2169999 marafivirus D Peach mosaic 183585 virus Peachrosette 65068 mosaic virus Peanut 35593 chlorotic streak virus Peanutclump 28355 virus Peanut yellow mosaic virus Pear blister 12783 cankerviroid Peaton 2560627 orthobunyavirus Peatvirus 2560629 peat2 Pecanmosaic- 1856031 associated virus Pecentumvirus 40523 A511 Penicillum2734569 brevicompactum polymycovirus 1 Pennisetum 221262 mosaic virusPepino mosaic virus[3] Pepo aphid- 1462681 borne yellows virus Pepperchat 574040 fruit viroid Pepper 2734493 chlorotic spot orthotospovirusPhietavirus X2 320850 Phifelvirus 1633149 FL1 Phikmvvirus 2733349 15pyoPhlox virus S 436066 Phnom Penh 64894 bat virus Phocid 47418alphaherpes- virus 1 Phocid 47419 gammaherpes virus 2 Phocid 2560643gammaherpes virus 3 Phocine 11240 morbillivirus Pholetesor ornigisbracovirus Phthorimaea 192584 operculella granulovirus Phutvirus 2733655PPpW4 Phyllosphere sclerotimonavirus Physalis 72539 mottle virusPhysarum polycephalum Tpl virus Phytophthora 310750 alphaendorna- virus1 Picardvirus 2734264 picard Pidgey 2509390 pidchovirus Piedvirus2733947 IMEDE1 Pienvirus 2733373 R801 Pifdecavirus 2733657 IBBPF7A Plumbark 675077 necrosis stem pitting- associated virus Plum pox 12211 virusPlumeria 1501716 mosaic virus Plutella 98383 xylostella granulovirus Poasemilatent 12328 virus Poaceae 1985392 associated gemycircular- virus 1Podivirus 2733948 S05C243 Poecivirus A 2560644 Pogseptimavirus 2733996PG07 Pogseptimavirus 2733997 VspSw1 Poindextervirus 2734196 BL10Poindextervirus 2748760 rogue Poinsettia 305785 latent virus Poinsettia113553 mosaic virus Pokeweed 1220025 mosaic virus Pokrovskaiavirus2733374 fHeYen301 Pokrovskaiavirus 2733375 pv8018 Polar bearmastadenovirus A Pollockvirus 2170215 pollock Pollyceevirus 2560679pollyC Polybotosvirus 2560286 Atuph07 Polygonum 430606 ringspotorthotospovirus Pomona bat 2049933 hepatitis B virus Pongine 159603gammaherpes virus 2 Poplar mosaic 12166 virus Popoffvirus 2560283 pv56Porcine 1985393 associated gemycircular- virus 1 Potato virus Y 12216Potato yellow 2230887 blotch virus Potato yellow 223307 mosaic Panamavirus Potato yellow 10827 mosaic virus Potato yellow 103881 vein virusPothos latent 44562 virus Potosi 2560646 orthobunyavirus Poushouvirus2560396 Poushou Pouzolzia 1225069 golden mosaic virus Primate T- 194443lymphotropic virus 3 Primolicivirus 2011081 Pf1 Primula 1511840malacoides virus 1 Priunavirus 2560652 PR1 Privet ringspot 2169960 virusProchlorococcus virus PHM1 Prospect Hill 1980485 orthohantavirusProtapanteles paleacritae bracovirus Providence 213633 virus Prune dwarf33760 virus Prunus latent 2560653 virus Prunus 37733 necrotic ringspotvirus Przondovirus 2733672 KN31 Pseudomonas 462590 virus YuaPseudoplusia includens virus Pseudotevenvirus 329381 RB16Pseudotevenvirus 115991 RB43 Psimunavirus 2734265 psiM2Psipapillomavirus 1 1177762 Psipapillomavirus 2 2170170Psipapillomavirus 3 2170171 Psittacid 50294 alphaherpesvirus 1Psittacine 2003673 atadenovirus A Psittacine 2169709 aviadenovirus BPsittacine 2734577 aviadenovirus C Psittacinepox 2169712 virusPteridovirus 2734351 filicis Pteridovirus 2734352 maydis Pteropodid2560693 alphaherpesvirus 1 Pteropox virus 1873698 Pteropus 1985395associated gemycircularvirus 1 Pteropus 1985404 associatedgemycircularvirus 10 Ptyasnivirus 1 2734501 Pukovnikvirus 540068pukovnik Pulverervirus 2170091 PFR1 Puma lentivirus 12804 Pumpkin2518373 polerovirus Pumpkin yellow 1410062 mosaic virus Punavirus P110678 Punavirus RCS47 2560452 Punavirus SJ46 2560732 Punique 2734468phlebovirus Punta Toro 1933186 phlebovirus Puumala 1980486orthohantavirus Pyrobaculum 1805492 filamentous virus 1 Pyrobaculum270161 spherical virus Qadamvirus 2733953 SB28 Qalyub 1980527orthonairovirus Qingdao virus J21 2734135 Qingling 2560694orthophasmavirus Quail pea mosaic virus Quailpox virus 400570Quaranjavirus 688437 johnstonense Quaranjavirus 688436 quaranfilenseQubevirus durum 39803 Qubevirus 39804 faecium Quezon 2501382 mobatvirusQuhwahvirus 2283289 kaihaidragon Quhwahvirus 2201441 ouhwah Quhwahvirus2182400 paschalis Rabbit associated 1985420 gemykroznavirus 1 Rabbitfibroma 10271 virus Rabbit 11976 hemorrhagic disease virus Rabovirus A1603962 Rabovirus B 2560695 Rabovirus C 2560696 Rabovirus D 2560697Raccoonpox 10256 virus Radish leaf curl 435646 virus Radish mosaic328061 virus Radish yellow 319460 edge virus Rafivirus A Rafivirus B2560699 Rafivirus C Raleigh virus 2734266 darolandstone Raleigh virus2734267 raleigh Ramie mosaic 1874886 Yunnan virus Ranid 85655herpesvirus 1 Ranid 389214 herpesvirus 2 Ranid 1987509 herpesvirus 3Ranunculus leaf 341110 distortion virus Ranunculus mild 341111 mosaicvirus Ranunculus 341112 mosaic virus Raptor 691961 siadenovirus ARaspberry bushy 12451 dwarf virus Raspberry leaf 326941 mottle virusRaspberry 12809 ringspot virus Rat associated 1985405 gemycircularvirus1 Rat associated 2170126 porprismacovirus 1 Rattail cactus 1123754necrosis- associated virus Rattus norvegicus 1679933 polyomavirus 1Rauchvirus BPP1 194699 Raven circovirus 345250 Ravin virus N15 40631Recovirus A 2560702 Red clover associated luteovirus Red clover 1323524cryptic virus 2 Red clover mottle 12262 virus Red clover 12267 necroticmosaic virus Red clover vein 590403 mosaic virus Red deerpox virusRedspotted 43763 grouper nervous necrosis virus Reginaelenavirus 2734071rv3LV2017 Rehmannia 425279 mosaic virus Rehmannia virus 1 2316740Reptilian 122203 ferlavirus Reptilian 226613 orthoreovirus Rerduovirus1982376 RER2 Rerduovirus 1109716 RGL3 Restivirus RSS1 2011075 Restonebolavirus 186539 Reticuloendo- 11636 theliosis virus Reyvirus rey1983751 Rhesus macaque 2170199 simian foamy virus Rhinolophus 2004965associated gemykibivirus 1 Rhinolophus 2004966 associated gemykibivirus2 Rhinolophus bat 693998 coronavirus HKU2 Rhinolophus 2501926ferrumequinum alphacoronavirus HuB-2013 Rhinovirus A 147711 Rhinovirus B147712 Rhinovirus C 463676 Rhizidiomyces virus Rhizoctonia 1408133cerealis alphaendornavirus 1 Rhizoctonia 2560704 magoulivirus 1 Sabo2560716 orthobunyavirus Saboya virus 64284 Sacbrood virus 89463Saccharomyces 186772 20S RNA narnavirus Saccharum streak 683179 virusSaclayvirus 2734138 Aci011 Saclayvirus 2734139 Aci022 Saclayvirus2734137 Aci05 Saetivirus fs2 1977306 Saetivirus VFJ 1977307 Saffronlatent 2070152 virus Saguaro cactus 52274 virus Saguinine 2169901gammaherpesvirus 1 Saikungvirus 2169924 HK633 Saikungvirus 2169925 HK75Saimiri sciureus 1236410 polyomavirus 1 Saimiriine 10353alphaherpesvirus 1 Saimiriine 1535247 betaherpesvirus 4 Saimiriine 10381gammaherpesvirus 2 Saint Floris phlebovirus Saint Louis 11080encephalitis virus Saint Valerien virus Sakhalin 1980528 orthonairovirusSakobuvirus A 1659771 Sal Vieja virus 64301 Salacisavirus 2734140 pssm2Salanga 2734471 phlebovirus Salasvirus phi29 10756 Salchichonvirus298338 LP65 Salehabad 1933188 phlebovirus Salem salemvirus 2560718Salivirus A 1330524 Salmo 2749930 aquapar amyxovirus Salmon gillpox2734576 virus Saphexavirus 1982380 VD13 Sapporo virus 95342 Sarcochilusvirus 104393 Y Sashavirus sasha 2734275 Sasquatchvirus 2734143 Y3Sasvirus BFK20 2560392 Satsuma dwarf 47416 virus Sauletekiovirus 2734030AAS23 Saumarez Reef 40012 virus Saundersvirus 2170234 Tp84 Sauropus leaf1130981 curl virus Sawgrhavirus 2734397 connecticut Sawgrhavirus 2734398longisland Sawgrhavirus 2734399 minto Sawgrhavirus 2734400 sawgrassScale drop 1697349 disease virus Scallion mosaic 157018 virus Scapularis2734431 ixovirus Scapunavirus 2560792 scapl Scheffersomyces 1300323segobiensis virus L Schefflera 2169729 ringspot virus Schiekvirus2560422 EFDG1 Schiekvirus 2734044 EFP01 Schiekvirus 2734045 EfV12Schistocerca gregaria entomopoxvirus Saphexavirus 1982380 VD13 Sophorayellow 2169837 stunt alphasatellite 5 Sorex araneus 2734504 coronavirusT14 Sorex araneus 2560769 polyomavirus 1 Sorex coronatus 2560770polyomavirus 1 Sorex minutus 2560771 polyomavirus 1 Sorghum 107804chlorotic spot virus Sorghum mosaic 32619 virus Sororoca 2560772orthobunyavirus Sortsnevirus 2734190 IME279 Sortsnevirus 2734189 sortsneSosuga 2560773 pararubulavirus Soupsvirus soups 1982563 Soupsvirus2560510 strosahl Soupsvirus wait 2560513 Souris 2169997 mammarenavirusSourvirus sour 2560509 South African 63723 cassava mosaic virus Southernbean 12139 mosaic virus Southern cowpea 196398 mosaic virus Southern1159195 elephant seal virus Southern rice 519497 black-streaked dwarfvirus Southern tomato 591166 virus Sowbane mosaic 378833 virus Soybean1985413 associated gemycircularvirus 1 Sophora yellow 2169837 stuntalphasatellite 5 Sorex araneus 2734504 coronavirus T14 Sorex araneus2560769 polyomavirus 1 Sorex coronatus 2560770 polyomavirus 1 Sorexminutus 2560771 polyomavirus 1 Sorghum 107804 chlorotic spot virusSorghum mosaic 32619 virus Sororoca 2560772 orthobunyavirus Sortsnevirus2734190 IME279 Switchgrass 2049938 mosaic- associated virus SymapivirusA Synechococcus 2734100 virus SRIM12-08 Synedrella leaf 1544378 curlalphasatellite Synedrella 1914900 yellow vein clearing virus Synetaeristenuifemur ichnovirus Syngnathid 2734305 ichthamaparvovirus 1 Synodus2749934 synodonvirus Tabernariusvirus 2560691 tabernarius Tacaiuma611707 orthobunyavirus Tacaribe 11631 mammarenavirus Tacheng 2734606uukuvirus Tahyna 2560796 orthobunyavirus Tangaroavirus 2733962 tv951510aTankvirus tank 1982567 Tapara 2734474 phlebovirus Tapirape 2560798pacuvirus Tapwovirus cesti 2509383 Taranisvirus 2734146 taranis Tarobacilliform 1634914 CH virus Taro bacilliform 178354 virus Tarumizu2734340 coltivirus Tataguine 2560799 orthobunyavirus Taterapox virus28871 Taupapillomavirus 1 1176148 Taupapillomavirus 2 1513274Taupapillomavirus 3 1961786 Taupapillomavirus 4 2170222 Taura syndrome142102 virus Tawavirus JSF7 2733965 Tea plant 2419939 necrotic ringblotch virus Tefnutvirus 2734147 siom18 Tegunavirus r1rt 1921705Tegunavirus 1921706 yenmtg1 Tehran 2734475 phlebovirus Telfairia golden2169737 mosaic virus Telfairia mosaic 1859135 virus Tellina virus 359995Tellina virus 1 321302 Telosma mosaic 400394 virus Tembusu virus 64293Tensaw 2560800 orthobunyavirus Tent-making bat 1508712 hepatitis B virusTeseptimavirus 2733885 YpsPG Testudine orthoreovirus Testudinid 2560801alphaherpesvirus 3 Tete 35319 orthobunyavirus Tetterwort vein 1712389chlorosis virus Teviot 2560803 pararubulavirus Thailand 1980492orthohantavirus Thalassavirus 2060093 thalassa Thaumasvirus 2734148stim4 Thermoproteus 292639 tenax spherical virus 1 Thermoproteus 10479tenax virus 1 Thermus virus 1714273 IN93 Thermus virus 1714272 P23-77Thetaarterivirus 2501999 kafuba Thetaarterivirus 2502000 mikelba lThetapapilloma- 197772 virus 1 Thetapolyomavirus 1891755 censtriataThetapolyomavirus 2218588 trebernacchii Thetapolyomavirus 2170103trepennellii Thetisvirus ssm1 2734149 Thiafora 1980529 orthonairovirusThimiri 1819305 orthobunyavirus Thin paspalum 1352511 asymptomatic virusThistle mottle virus Thogotovirus 11318 dhoriense Thogotovirus 11569thogotoense Thomixvirus 2560804 OH3 Thornevirus 2560336 SP15 Thoseaasigna 83810 virus Thottopalayam 2501370 thottimvirus Thunberg 299200fritillary mosaic virus Thysanoplusia 101850 orichalcea nucleopolyhedrovirus Tiamatvirus 268748 PSSP7 Tibetan frog 2169919 hepatitis B virusTibrovirus 1987018 alphaekpoma Tibrovirus 2170224 beatrice Tibrovirus1987019 betaekpoma Tibrovirus 1972586 coastal Tibrovirus congo 1987017Tibrovirus 1987013 sweetwater Tibrovirus 1972584 tibrogargan Tickassociated 2560805 circovirus 1 Tick associated 2560806 circovirus 2Tick-borne 11084 encephalitis virus Tico phebovirus 2734476 Tidunavirus2560834 pTD1 Tidunavirus 2560833 VP4B Tiger puffer 43764 nervousnecrosis virus Tigray 2560807 orthohantavirus Tigrvirus E122 431892Tigrvirus E202 431893 Tobacco leaf curl 439423 Comoros virus Tobaccoleaf curl 336987 Cuba virus Tobacco leaf curl 2528965 Dominican Republicvirus Tobacco leaf curl 2010326 Japan betasatellite Tobacco leaf curl2010327 Patna betasatellite Tobacco leaf curl 905054 Pusa virus Tobaccoleaf curl 409287 Thailand virus Tobacco leaf curl 211866 Yunnan virusTobacco leaf curl 223337 Zimbabwe virus Tobacco leaf 196691 rugose virusVeracruzvirus 1032892 heldan Veracruzvirus 2003502 rockstar Verbenalatent 134374 virus Verbena virus Y 515446 Vernonia crinkle 1925153virus Vernonia yellow 666635 vein betasatellite Vernonia yellow 2169908vein Fujian alphasatellite Vernonia yellow 2050589 vein Fujianbetasatellite Vernonia yellow 1001341 vein Fujian virus Vernonia yellow367061 vein virus Versovirus 2011076 VfO3K6 Verticillium 759389 dahliaechrysovirus 1 Vesicular 35612 exanthema of swine virus Vesiculovirus1972579 alagoas Vesiculovirus 1972567 bogdanovac Whitefly- 2169744associated begomovirus 7 White-tufted-ear 2170205 marmoset simian foamyvirus Whitewater 46919 Arroyo mammarenavirus Wifcevirus 2734154 ECML117Wifcevirus 2734155 FEC19 Wifcevirus WFC 2734156 Wifcevirus WFH 2734157Wigeon 1159908 coronavirus HKU20 Wild cucumber 70824 mosaic virus Wildmelon banding virus Wild onion 1862127 symptomless virus Wild potato187977 mosaic virus Wild tomato 400396 mosaic virus Wild Vitis latent2560839 virus Wilnyevirus 2560486 billnye Wilsonroadvirus 2734007 Sd1Winged bean 2169693 alphaendornavirus 1 Winklervirus 2560752 chi14Wiseana signata 65124 nucleopolyhedro virus Wissadula golden 51673mosaic virus Wissadula yellow 1904884 mosaic virus Wisteria 1973265badnavirus 1 Wisteria vein 201862 mosaic virus Witwatersrand 2560841orthobunyavirus Wizardvirus 2170253 twister6 Wizardvirus 2170254 wizardWoesvirus woes 1982751 Wolkberg 2170059 orthobunyavirus Wongorr virus47465 Wongtaivirus 2169922 HK542 Woodchuck 35269 hepatitis virusWoodruffvirus 1982746 TP1604 Woodruffvirus 1982747 YDN12 Woolly monkey68416 hepatitis B virus Woolly monkey 11970 sarcoma virus Wound tumor10987 virus Wphvirus 2560329 BPS10C Wphvirus BPS13 1987727 Wphvirushakuna 1987729 Wphvirus 1987728 megatron Wphvirus WPh 1922328 Wuchang1980542 cockroach orthophasmavirus 1 Wuhan mivirus 2507319 Wuhanmosquito 1980543 orthophasmavirus 1 Wuhan mosquito 1980544orthophasmavirus 2 Wuhan virus 2733969 PHB01 Wuhanvirus 2733970 PHB02Wumivirus 2509286 millepedae Wumpquatrovirus 400567 WMP4 Wumptrevirus440250 WMP3 Wutai mosquito 1980612 phasivirus Wyeomyia 273350orthobunyavirus Xanthophyllomyces 1167690 dendrorhous virus L1AXanthophyllomyces 1167691 dendrorhous virus L1B Xapuri 2734417mammarenavirus Xestia c-nigrum 51677 granulovirus Xiamenvirus 1982373RDJL1 Xiamenvirus 1982374 RDJL2 Xilang striavirus 2560844 Xinzhoumivirus 2507320 Xipapillomavirus 1 10561 Xipapillomavirus 2 1513273Yokohamavirus 1980942 PEi21 Yokose virus 64294 Yoloswagvirus 2734158yoloswag Yongjia 2734607 uukuvirus Youcai mosaic 228578 virus Yunnanorbivirus 306276 Yushanvirus 2733978 Spp001 Yushanvirus 2733979 SppYZU05Yuyuevirus 2508254 beihaiense Yuyuevirus 2508255 shaheense Zaireebolavirus 186538 Zaliv Terpeniya 2734608 uukuvirus Zantedeschia 270478mild mosaic virus Zarhavirus 2734410 zahedan Zika virus 64320

The cascade assays described herein are particularly well-suited forsimultaneous testing of multiple targets. Pools of two to 10,000 targetnucleic acids of interest may be employed, e.g., pools of 2-1000, 2-100,2-50, or 2-10 target nucleic acids of interest. Further testing may beused to identify the specific member of the pool, if warranted.

While the methods described herein do not require the target nucleicacid of interest to be DNA (and in fact it is specifically contemplatedthat the target nucleic acid of interest may be RNA), it is understoodby those in the field that a reverse transcription step to converttarget RNA to cDNA may be performed prior to or while contacting thebiological sample with the composition.

Nucleic Acid-Guided Nucleases

The cascade assays comprise nucleic acid-guided nucleases in thereaction mix, either provided as a protein, a coding sequence for theprotein, or, in many embodiments, in a ribonucleoprotein (RNP) complex.In some embodiments, the one or more nucleic acid-guided nucleases inthe reaction mix may be, for example, a Cas nucleic acid-guidednuclease. Any nucleic acid-guided nuclease having both cis- andtrans-cleavage activity may be employed, and the same nucleicacid-guided nuclease may be used for both RNP complexes or differentnucleic acid-guided nucleases may be used in RNP1 and RNP2. For example,RNP1 and RNP2 may both comprise Cas12a nucleic acid-guided nucleases, orRNP1 may comprise a Cas13 nucleic acid-guided nuclease and RNP2 maycomprise a Cas12a nucleic acid-guided nuclease or vice versa. Inembodiments where a variant nucleic acid-guided nuclease is employed,only RNP2 will comprise the variant, and RNP1 may comprise either aCas12a or Cas13 nucleic acid-guided nuclease. In embodiments where avariant nucleic acid-guided nuclease is not employed, either or bothRNP1 and RNP2 can comprise a Cas13 nucleic acid-guided nuclease. Notethat trans-cleavage activity is not triggered unless and untilcis-cleavage activity (i.e., sequence specific activity) is initiated.Nucleic acid-guided nucleases include Type V and Type VI nucleicacid-guided nucleases, as well as nucleic acid-guided nucleases thatcomprise a RuvC nuclease domain or a RuvC-like nuclease domain but lackan HNH nuclease domain. Nucleic acid-guided nucleases with theseproperties are reviewed in Makarova and Koonin, Methods Mol. Biol.,1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology,37:67-78 (2020) and updated databases of nucleic acid-guided nucleasesand nuclease systems that include newly-discovered systems includeBioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org);Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder(crispercas.i2bc.paris-saclay.fr).

The type of nucleic acid-guided nuclease utilized in the method ofdetection depends on the type of target nucleic acid of interest to bedetected. For example, a DNA nucleic acid-guided nuclease (e.g., aCas12a, Cas14a, or Cas3) should be utilized if the target nucleic acidof interest is a DNA molecule, and an RNA nucleic acid-guided nuclease(e.g., Cas13a or Cas12g) should be utilized if the target nucleic acidof interest is an RNA molecule. Exemplary nucleic acid-guided nucleasesinclude, but are not limited to, Cas RNA-guided DNA nucleic acid-guidednucleases, such as Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b,Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, and Cas12j; CasRNA-guided RNA nucleic acid-guided nucleases, such as Cas13a (LbaCas13,LbuCas13, LwaCas13), Cas13b (e.g., CccaCas13b, PsmCas13b), and Cas12g;and any other nucleic acid (DNA, RNA, or cDNA) targeting nucleicacid-guided nuclease with cis-cleavage activity and collateraltrans-cleavage activity. In some embodiments, the nucleic acid-guidednuclease is a Type V CRISPR-Cas nuclease, such as Cas12a, Cas13a, orCas14a. In some embodiments, the nucleic acid-guided nuclease is a TypeI CRISPR-Cas nuclease, such as Cas3. Type II and Type VI nucleicacid-guided nucleases may also be employed.

In an RNP with a single crRNA (i.e., lacking/without a tracrRNA), Cas12anucleases and related homologs and orthologs interact with a PAM(protospacer adjacent motif) sequence in a target nucleic acid for dsDNAunwinding and R-loop formation. Cas12a nucleases employ a multistepmechanism to ensure accurate recognition of spacer sequences in thetarget nucleic acid. The WED, REC1 and PAM-interacting (PI) domains ofCas12a nucleases are responsible for PAM recognition and for initiatinginvasion of the crRNA in the target dsDNA and for R-loop formation. Ithas been hypothesized that a conserved lysine residue is inserted intothe dsDNA duplex, possibly initiating template strand/non-templatestrand unwinding. (See Jinek, et al, Mol. Cell, 73(3):589-600.e4(2019).) PAM binding further introduces a kink in the target strand,which further contributes to local strand separation and facilitatesbase paring of the target strand to the seed segment of the crRNA whilethe displaced non-target strand is stabilized by interactions with thePAM-interacting domains. (Id.) The variant nucleic acid-guided nucleasesdisclosed herein and discussed in detail below have been engineered todisrupt one or both of the WED and PI domains to reconfigure the site ofunwinding and R-loop formation to, e.g., sterically obstruct dsDNAtarget nucleic acids from binding to the variant nucleic acid-guidednuclease and/or to minimize strand separation and/or stabilization ofthe non-target strand. Though contrary to common wisdom, engineering thevariant nucleic acid-guided nucleases in this way contributes to arobust and high-fidelity cascade assay.

The variant nucleic acid-guided nucleases disclosed herein are variantsof wildtype Type V nucleases LbCas12a (Lachnospriaceae bacteriumCas12a), AsCas 12a (Acidaminococcus sp. BV3L6 Cas12a), CtCas12a(Candidatus Methanoplasma termitum Cas12a), EeCas12a (Eubacteriumeligens Cas12a), Mb3Cas12a (Moraxella bovoculi Cas12a), FnCas12a(Francisella novicida Cas12a), FnoCas12a (Francisella tularensis subsp.novicida FTG Cas12a), FbCas 12a (Flavobacteriales bacterium Cas12a),Lb4Cas 12a (Lachnospira eligens Cas12a), MbCas12a (Moraxella bovoculiCas12a), Pb2Cas12a (Prevotella bryantii Cas12a), PgCas12a (CandidatusParcubacteria bacterium Cas12a), AaCas12a (Acidaminococcus sp. Cas12a),BoCas 12a (Bacteroidetes bacterium Cas12a), CMaCas 12a (CandidatusMethanomethylophilus alvus CMx1201 Cas12a), and to-be-discoveredequivalent Cas12a nucleic acid-guided nucleases and homologs andorthologs of these nucleic acid-guided nucleases (and other nucleicacid-guided nucleases that exhibit both cis-cleavage and trans-cleavageactivity), where mutations have been made to the PAM interacting domainssuch that double-stranded DNA (dsDNA) substrates are bound much moreslowly to the variant nucleic acid-guided nucleases than to theirwildtype nucleic acid-guided nuclease counterpart, yet single-strandedDNA (ssDNA) substrates are bound at the same rate or nearly so as theirwildtype nucleic acid-guided nuclease counterpart. The variant nucleicacid-guided nucleases comprise reconfigured domains that interact withthe PAM region or surrounding sequences on the blocked nucleic acidmolecules to achieve this phenotype and are described in detail below.

Guide RNA (gRNA)

The present disclosure detects a target nucleic acid of interest via areaction mixture containing at least two guide RNAs (gRNAs) eachincorporated into a different RNP complex (i.e., RNP1 and RNP2).Suitable gRNAs include at least one crRNA region to enable specificityin every reaction. The gRNA of RNP1 is specific to a target nucleic acidof interest and the gRNA of RNP2 is specific to an unblocked nucleicacid or a synthesized activating molecule (both described in detailbelow). As will be clear given the description below, an advantageousfeature of the cascade assay is that, with the exception of the gRNA inthe RNP1 (i.e., the gRNA specific to the target nucleic acid ofinterest), the cascade assay components can stay the same (i.e., areidentical or substantially identical) no matter what target nucleicacid(s) of interest are being detected, and the gRNA in RNP1 is easilyreprogrammable.

Like the nucleic acid-guided nuclease, the gRNA may be provided in thecascade assay reaction mix in a preassembled RNP, as an RNA molecule, ormay also be provided as a DNA sequence to be transcribed, in, e.g., avector backbone. Providing the gRNA in a pre-assembled RNP complex(i.e., RNP1 or RNP2) is preferred if rapid kinetics are preferred. Ifprovided as a gRNA molecule, the gRNA sequence may include multipleendoribonuclease recognition sites (e.g., Csy4) for multiplexprocessing. Alternatively, if provided as a DNA sequence to betranscribed, an endoribonuclease recognition site may be encoded betweenneighboring gRNA sequences such that more than one gRNA can betranscribed in a single expression cassette. Direct repeats can alsoserve as endoribonuclease recognition sites for multiplex processing.Guide RNAs are generally about 20 nucleotides to about 300 nucleotidesin length and may contain a spacer sequence containing a plurality ofbases and complementary to a protospacer sequence in the targetsequence. The gRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%,95%, 97.5%, 98%, 99%, or more complementary to its intended targetnucleic acid of interest.

The gRNA of RNP1 is capable of complexing with the nucleic acid-guidednuclease of RNP1 to perform cis-cleavage of a target nucleic acid ofinterest (e.g., a DNA or RNA), which triggers non-sequence specifictrans-cleavage of other molecules in the reaction mix. Guide RNAsinclude any polynucleotide sequence having sufficient complementaritywith a target nucleic acid of interest (or target sequences generated byunblocking blocked nucleic acid molecules or target sequences generatedby synthesizing synthesized activating molecules as described below).Target nucleic acids of interest (describe in detail above) preferablyinclude a protospacer-adjacent motif (PAM), and, following gRNA binding,the nucleic acid-guided nuclease induces a double-stranded break eitherinside or outside the protospacer region of the target nucleic acid ofinterest.

In some embodiments, the gRNA (e.g., of RNP1) is an exo-resistantcircular molecule that can include several DNA bases between the 5′ endand the 3′ end of a natural guide RNA and is capable of binding a targetsequence. The length of the circularized guide for RNP1 can be such thatthe circular form of guide can be complexed with a nucleic acid-guidednuclease to form a modified RNP1 which can still retain its cis-cleavagei.e., (specific) and trans-cleavage (i.e., non-specific) nucleaseactivity.

In any of the foregoing embodiments, the gRNA may be a modified ornon-naturally occurring nucleic acid molecule. In some embodiments, thegRNAs of the disclosure may further contain a locked nucleic acid (LNA),a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). Byway of further example, a modified nucleic acid molecule may contain amodified or non-naturally occurring nucleoside, nucleotide, and/orinternucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modifiednucleoside, a 2′-fluoro (2′-F) modified nucleoside, and aphosphorothioate (PS) bond, or any other nucleic acid moleculemodifications described herein.

Ribonucleoprotein (RNP) Complex

As described above, although the cascade assay “reaction mix” maycomprise separate nucleic acid-guided nucleases and gRNAs (or codingsequences therefor), the cascade assays preferably comprise preassembledribonucleoprotein complexes (RNPs) in the reaction mix, allowing forfaster detection kinetics. The present cascade assay employs at leasttwo types of RNP complexes—RNP1 and RNP2—each type containing a nucleicacid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise the samenucleic acid-guided nuclease or may comprise different nucleicacid-guided nucleases; however, the gRNAs in RNP1 and RNP2 are differentand are configured to detect different nucleic acids. In someembodiments, the reaction mixture contains about 1 fM to about 10 μM ofa given RNP1, or about 1 pM to about 1 μM of a given RNP1, or about 10pM to about 500 pM of a given RNP1. In some embodiments the reactionmixture contains about 6×10⁴ to about 6×10¹² complexes per microliter(μl) of a given RNP1, or about 6×10⁶ to about 6×10¹⁰ complexes permicroliter (μl) of a given RNP1. In some embodiments, the reactionmixture contains about 1 fM to about 500 μM of a given RNP2, or about 1pM to about 250 μM of a given RNP2, or about 10 pM to about 100 μM of agiven RNP2. In some embodiments the reaction mixture contains about6×10⁴ to about 6×10¹² complexes per microliter (μl) of a given RNP2 orabout 6×10⁶ to about 6×10¹² complexes per microliter (μl) of a givenRNP2. See Example II below describing preassembling RNPs and Examples Vand VI below describing various cascade assay conditions where therelative concentrations of RNP2 and the blocked nucleic acid moleculesis adjusted as described below.

In any of the embodiments of the disclosure, the reaction mixtureincludes 1 to about 1,000 different RNP1s (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50,75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, or 1,0000 or more RNP1s), wheredifferent RNPls comprise a different gRNA (or crRNA thereof)polynucleotide sequence. For example, a reaction mixture designed forenvironmental or oncology testing comprises more than one uniqueRNP1-gRNA (or RNP1-crRNA) ribonucleoprotein complex for the purpose ofdetecting more than one target nucleic acid of interest. That is, morethan one RNP1 may also be present for the purpose of targeting onetarget nucleic acid of interest from many sources or for targeting morethan one target nucleic acid of interest from a single source.

In any of the foregoing embodiments, the gRNA of RNP1 may be homologousor heterologous, relative to the gRNA of other RNP1(s) present in thereaction mixture. A homologous mixture of RNP1 gRNAs has a number ofgRNAs with the same nucleotide sequence, whereas a heterologous mixtureof RNP1 gRNAs has multiple gRNAs with different nucleotide sequences(e.g., gRNAs targeting different loci, genes, variants, and/or microbialspecies). Therefore, the disclosed methods of identifying one or moretarget nucleic acids of interest may include a reaction mixturecontaining more than two heterologous gRNAs, more than threeheterologous gRNAs, more than four heterologous gRNAs, more than fiveheterologous gRNAs, more than six heterologous gRNAs, more than sevenheterologous gRNAs, more than eight heterologous gRNAs, more than nineheterologous gRNAs, more than ten heterologous gRNAs, more than elevenheterologous gRNAs, more than twelve heterologous gRNAs, more thanthirteen heterologous gRNAs, more than fourteen heterologous gRNAs, morethan fifteen heterologous gRNAs, more than sixteen heterologous gRNAs,more than seventeen heterologous gRNAs, more than eighteen heterologousgRNAs, more than nineteen heterologous gRNAs, more than twentyheterologous gRNAs, more than twenty-one heterologous gRNAs, more thantwenty-three heterologous gRNAs, more than twenty-four heterologousgRNAs, or more than twenty-five heterologous gRNAs. Such a heterologousmixture of RNP1 gRNAs in a single reaction enables multiplex testing.

As a first non-limiting example of a heterologous mixture of RNP1 gRNAs,the reaction mixture may contain: a number of RNPls (RNP1-1s) having agRNA targeting parainfluenza virus 1; a number of RNP1s (RNP1-2s) havinga gRNA targeting human metapneumovirus; a number of RNP1s (RNP1-3s)having a gRNA targeting human rhinovirus; a number of RNP1s (RNP1-4s)having a gRNA targeting human enterovirus; and a number of RNP1s(RNP1-5s) having a gRNA targeting coronavirus HKU1. As a secondnon-limiting example of a heterologous mixture of RNP1 gRNAs, thereaction mixture may contain: a number of RNPls containing a gRNAtargeting two or more SARS-Co-V-2 variants, e.g., B.1.1.7, B.1.351, P.1,B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 and subvariantsthereof.

As another non-limiting example of a heterologous mixture of RNP1 gRNAs,the reaction mixture may contain RNP1s targeting two or more targetnucleic acids of interest from organisms that infect grapevines, such asGuignardia bidwellii (RNP1-1), Uncinula necator (RNP1-2), Botrytiscincerea (RNP1-3), Plasmopara viticola (RNP1-4), and Botryotinisfuckleina (RNP1-5).

Reporter Moieties

The cascade assay detects a target nucleic acid of interest viadetection of a signal generated in the reaction mix by a reportermoiety. In some embodiments the detection of the target nucleic acid ofinterest occurs virtually instantaneously. For example, see the resultsreported in Example VI for assays comprising 3e4 or 30 copies of MRSAtarget and within 1 minute or less at 3 copies of MRSA target (see,e.g., FIGS. 10B-10H). Reporter moieties can comprise DNA, RNA, a chimeraof DNA and RNA, and can be single stranded, double stranded, or a moietythat is a combination of single stranded portions and double strandedportions.

Depending on the type of reporter moiety used, trans- and/orcis-cleavage by the nucleic acid-guided nuclease in RNP2 releases asignal. In some embodiments, trans-cleavage of stand-alone reportermoieties (e.g., not bound to any blocked nucleic acid molecules orblocked primer molecules) may generate signal changes at rates that areproportional to the cleavage rate, as new RNP2s are activated over time(shown in FIG. 1B and at top of FIG. 4 ). Trans-cleavage by either anactivated RNP1 or an activated RNP2 may release a signal. In alternativeembodiments and preferably, the reporter moiety may be bound to theblocked nucleic acid molecule, where trans-cleavage of the blockednucleic acid molecule (or blocked primer molecule) and conversion to anunblocked nucleic acid molecule (or unblocked primer molecule) maygenerate signal changes at rates that are proportional to the cleavagerate, as new RNP2s are activated over time, thus allowing for real timereporting of results (shown at FIG. 4 , center). In yet anotherembodiment, the reporter moiety may be bound to a blocked nucleic acidmolecule such that cis-cleavage following the binding of the RNP2 to anunblocked nucleic acid molecule releases a PAM distal sequence, which inturn generates a signal at rates that are proportional to the cleavagerate (shown at FIG. 4 , bottom). In this case, activation of RNP2 bycis- (target specific) cleavage of the unblocked nucleic acid moleculedirectly produces a signal, rather than producing a signal viaindiscriminate trans-cleavage activity. Alternatively or in addition, areporter moiety may be bound to the gRNA.

The reporter moiety may be a synthetic molecule linked or conjugated toa reporter and quencher such as, for example, a TaqMan probe with a dyelabel (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end.The reporter and quencher may be about 20-30 bases apart or less (i.e.,10-11 nm apart or less) for effective quenching via fluorescenceresonance energy transfer (FRET). Alternatively, signal generation mayoccur through different mechanisms. Other detectable moieties, labels,or reporters can also be used to detect a target nucleic acid ofinterest as described herein. Reporter moieties can be labeled in avariety of ways, including direct or indirect attachment of a detectablemoiety such as a fluorescent moiety, hapten, or colorimetric moiety.

Examples of detectable moieties include various radioactive moieties,enzymes, prosthetic groups, fluorescent markers, luminescent markers,bioluminescent markers, metal particles, and protein-protein bindingpairs, e.g., protein-antibody binding pairs. Examples of fluorescentmoieties include, but are not limited to, yellow fluorescent protein(YFP), green fluorescence protein (GFP), cyan fluorescence protein(CFP), umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansylchloride, phycocyanin, and phycoerythrin. Examples of bioluminescentmarkers include, but are not limited to, luciferase (e.g., bacterial,firefly, click beetle and the like), luciferin, and aequorin. Examplesof enzyme systems having visually detectable signals include, but arenot limited to, galactosidases, glucorinidases, phosphatases,peroxidases, and cholinesterases. Identifiable markers also includeradioactive elements such as ¹²⁵1, ³⁵S, ¹⁴C, or ³H. Reporters can alsoinclude a change in pH or charge of the cascade assay reaction mix.

The methods used to detect the generated signal will depend on thereporter moiety or moieties used. For example, a radioactive label canbe detected using a scintillation counter, photographic film as inautoradiography, or storage phosphor imaging. Fluorescent labels can bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence can bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Enzymatic labels can be detected byproviding the appropriate substrates for the enzyme and detecting theresulting reaction product. Simple colorimetric labels can be detectedby observing the color associated with the label. When pairs offluorophores are used in an assay, fluorophores are chosen that havedistinct emission patterns (wavelengths) so that they can be easilydistinguished. In some embodiments, the signal can be detected bylateral flow assays (LFAs). Lateral flow tests are simple devicesintended to detect the presence or absence of a target nucleic acid ofinterest in a sample. LFAs can use nucleic acid molecules conjugatednanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detectionprobe, which hybridizes to a complementary target sequence. (See FIG. 9and the description thereof below.) The classic example of an LFA is thehome pregnancy test.

Single-stranded, double-stranded or reporter moieties comprising bothsingle- and double-stranded portions can be introduced to show a signalchange proportional to the cleavage rate, which increases with every newactivated RNP2 complex over time. In some embodiments and as describedin detail below, reporter moieties can also be embedded into the blockednucleic acid molecules (or blocked primer molecules) for real timereporting of results.

For example, the method of detecting a target nucleic acid molecule in asample using a cascade assay as described herein can involve contactingthe reaction mix with a labeled detection ssDNA containing a fluorescentresonance energy transfer (FRET) pair, a quencher/phosphor pair, orboth. A FRET pair consists of a donor chromophore and an acceptorchromophore, where the acceptor chromophore may be a quencher molecule.FRET pairs (donor/acceptor) suitable for use include, but are notlimited to, EDANS/fluorescein, IAEDANS/fluorescein,fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL,fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5, TexasRed/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL, andfluorescein/LC Red 705. In addition, a fluorophore/quantum dotdonor/acceptor pair can be used. EDANS is(5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid);DABCYL is 4-(4- dimethylaminophenyl) diazenylbenzoic acid. Usefulquenchers include, but are not limited to, BHQ, DABCYL, QSY 7 and QSY33.

In any of the foregoing embodiments, the reporter moiety may compriseone or more modified nucleic acid molecules, containing a modifiednucleoside or nucleotide. In some embodiments the modified nucleoside ornucleotide is chosen from 2′-O-methyl (2′-O-Me) modified nucleoside, a2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond,or any other nucleic acid molecule modifications described below.

Nucleic Acid Modifications

For any of the nucleic acid molecules described herein (e.g., blockednucleic acid molecules, blocked primer molecules, gRNAs, templatemolecules, synthesized activating molecules, and reporter moieties), thenucleic acid molecules may be used in a wholly or partially modifiedform. Typically, modifications to the blocked nucleic acid molecules,gRNAs, template molecules, reporter moieties, and blocked primermolecules described herein are introduced to optimize the molecule'sbiophysical properties (e.g., increasing nucleic acid-guided nucleaseresistance and/or increasing thermal stability). Modifications typicallyare achieved by the incorporation of, for example, one or morealternative nucleosides, alternative sugar moieties, and/or alternativeinternucleoside linkages.

For example, one or more of the cascade assay components may include oneor more of the following nucleoside modifications: 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynylderivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The nucleicacid molecules described herein (e.g., blocked nucleic acid molecules,blocked primer molecules, gRNAs, reporter molecules, synthesizedactivating molecules, and template molecules) may also includenucleobases in which the purine or pyrimidine base is replaced withother heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine, and/or 2-pyridone. Further modification of the nucleicacid molecules described herein may include nucleobases disclosed inU.S. Pat. No. 3,687,808; Kroschwitz, ed., The Concise Encyclopedia ofPolymer Science and Engineering, New York, John Wiley & Sons, 1990, pp.858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); andSanghvi, Chapter 16, Antisense Research and Applications, CRC Press,Gait, ed., 1993, pp. 289-302.

In addition to or as an alternative to nucleoside modifications, thecascade assay components may comprise 2′ sugar modifications, including2′-O-methyl (2′ -O-Me), 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and/or2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂OCH₂N(CH₃)₂. Other possible 2′-modifications that can modify thenucleic acid molecules described herein (i.e., blocked nucleic acidmolecules, gRNAs, synthesized activating molecules, reporter molecules,and blocked primer molecules) may include all possible orientations ofOH; F; O-, S-, or N-alkyl (mono- or di-); O-, S-, or N-alkenyl (mono- ordi-); O-, S- or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, whereinthe alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 toC10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugarsubstituent groups include, e.g., aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl(—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugarsubstituent groups may be in the arabino (up) position or ribo (down)position. In some embodiments, the 2′-arabino modification is 2′-F.Similar modifications may also be made at other positions on theinterfering RNA molecule, particularly the 3′ position of the sugar onthe 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the5′ position of 5′ terminal nucleotide. Oligonucleotides may also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar.

Finally, modifications to the cascade assay components may compriseinternucleoside modifications such as phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates, and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.

The Signal Boosting Cascade Assay Employing Blocked Nucleic AcidMolecules

Before getting to the details relating to addressing undesired unwindingof the blocked nucleic acid molecules (or blocked primer molecules),understanding the cascade assay itself is key. FIG. 1B, described above,depicts the cascade assay generally. A specific embodiment of thecascade assay utilizing blocked nucleic acid molecules is depicted inFIG. 2A and described in detail below. In this embodiment, a blockednucleic acid is used to prevent the activation of RNP2 in the absence ofa target nucleic acid of interest. The method in FIG. 2A begins withproviding the cascade assay components RNP1 (201), RNP2 (202) andblocked nucleic acid molecules (203). RNP1 (201) comprises a gRNAspecific for a target nucleic acid of interest and a nucleic acid-guidednuclease (e.g., Cas 12a or Cas 14 for a DNA target nucleic acid ofinterest or a Cas 13a for an RNA target nucleic acid of interest) andRNP2 (202) comprises a gRNA specific for an unblocked nucleic acidmolecule and a nucleic acid-guided nuclease (again, e.g., Cas 12a or Cas14 for a DNA unblocked nucleic acid molecule or a Cas 13a for an RNAunblocked nucleic acid molecule). As described above, the nucleicacid-guided nucleases in RNP1 (201) and RNP2 (202) can be the same ordifferent depending on the type of target nucleic acid of interest andunblocked nucleic acid molecule. What is key, however, is that thenucleic acid-guided nucleases in RNP1 and RNP2 may be activated to havetrans-cleavage activity following initiation of cis-cleavage activity.

In a first step, a sample comprising a target nucleic acid of interest(204) is added to the cascade assay reaction mix. The target nucleicacid of interest (204) combines with and activates RNP1 (205) but doesnot interact with or activate RNP2 (202). Once activated, RNP1 binds thetarget nucleic acid of interest (204) and cuts the target nucleic acidof interest (204) via sequence-specific cis-cleavage, activatingnon-specific trans-cleavage of other nucleic acids present in thereaction mix, including the blocked nucleic acid molecules (203). Atleast one of the blocked nucleic acid molecules (203) becomes anunblocked nucleic acid molecule (206) when the blocking moiety (207) isremoved. As described below, “blocking moiety” may refer to nucleosidemodifications, topographical configurations such as secondarystructures, and/or structural modifications.

Once at least one of the blocked nucleic acid molecules (203) isunblocked, the unblocked nucleic acid molecule (206) can then bind toand activate an RNP2 (208). Because the nucleic acid-guided nucleases inthe RNP1s (205) and RNP2s (208) have both cis- and trans-cleavageactivity, the trans-cleavage activity causes more blocked nucleic acidmolecules (203) become unblocked nucleic acid molecules (206) triggeringactivation of even more RNP2s (208) and more trans-cleavage activity ina cascade. FIG. 2A at bottom depicts the concurrent activation ofreporter moieties. Intact reporter moieties (209) comprise a quencher(210) and a fluorophore (211) linked by a nucleic acid sequence. Asdescribed above in relation to FIG. 1B, the reporter moieties are alsosubject to trans-cleavage by activated RNP1 (205) and RNP2 (208). Theintact reporter moieties (209) become activated reporter moieties (212)when the quencher (210) is separated from the fluorophore (211),emitting a fluorescent signal (213). Signal strength increases rapidlyas more blocked nucleic acid molecules (203) become unblocked nucleicacid molecules (206) triggering cis-cleavage activity of more RNP2s(208) and thus more trans-cleavage activity of the reporter moieties(209). Again, the reporter moieties are shown here as separate moleculesfrom the blocked nucleic acid molecules, but other configurations may beemployed and are discussed in relation to FIG. 4 . One particularlyadvantageous feature of the cascade assay is that, with the exception ofthe gRNA in the RNP1 (gRNA1), the cascade assay components are modularin the sense that the components stay the same no matter what targetnucleic acid(s) of interest are being detected.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule(220) and an exemplary technique for unblocking the blocked nucleic acidmolecules described herein. A blocked single-stranded ordouble-stranded, circular or linear, DNA or RNA molecule (220)comprising a target strand (222) may contain a partial hybridizationwith a complementary non-target strand nucleic acid molecule (224)containing unhybridized and cleavable secondary loop structures (226)(e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissinghairpins, internal loops, bulges, and multibranch loops). Trans-cleavageof the loops by, e.g., activated RNP1s or RNP2s, generates short strandnucleotide sequences or regions (228) which, because of the short lengthand low melting temperature T_(m) can dehybridize at room temperature(e.g., 15°-25° C.), thereby unblocking the blocked nucleic acid molecule(220) to create an unblocked nucleic acid molecule (230), enabling theinternalization of the unblocked nucleic acid molecule (230) (targetstrand) into an RNP2, leading to RNP2 activation.

A blocked nucleic acid molecule may be single-stranded ordouble-stranded, circular or linear, and may further contain a partiallyhybridized nucleic acid sequence containing cleavable secondary loopstructures, as exemplified by “L” in FIGS. 2C-2E. Such blocked nucleicacid molecules typically have a low binding affinity, or highdissociation constant (K_(d)) in relation to binding to RNP2 and may bereferred to herein as a high K_(d) nucleic acid molecule. In the contextof the present disclosure, the binding of blocked or unblocked nucleicacid molecules or blocked or unblocked primer molecules to RNP2, lowK_(d) values range from about 100 fM to about 1 aM or lower (e.g., 100zM) and high K_(d) values are in the range of 100 nM to about 10-100 10mM and thus are about 10⁵-, 10⁶-, 10⁷-, 10⁸-, 10⁹- to 10¹⁰-fold orhigher as compared to low K_(d) values. Of course, the ideal blockednucleic acid molecule would have an “infinite K_(d).”

The blocked nucleic acid molecules (high K_(d) molecules) describedherein can be converted into unblocked nucleic acid molecules (low K_(d)molecules—also in relation to binding to RNP2) via cleavage ofnuclease-cleavable regions (e.g., via active RNP1s and RNP2s). Theunblocked nucleic acid molecule has a higher binding affinity for thegRNA in RNP2 than does the blocked nucleic acid molecule, although, asdescribed below, there is some “leakiness” where some blocked nucleicacid molecules are able to interact with the gRNA in the RNP2 triggeringundesired unwinding.

Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2activation triggers trans-cleavage activity, which in turn leads to moreRNP2 activation by further cleaving blocked nucleic acid molecules,resulting in a positive feedback loop or cascade.

In embodiments where blocked nucleic acid molecules are linear and/orform a secondary structure, the blocked nucleic acid molecules may besingle-stranded (ss) or double-stranded (ds) and contain a firstnucleotide sequence and a second nucleotide sequence. The firstnucleotide sequence has sufficient complementarity to hybridize to agRNA of RNP2, and the second nucleotide sequence does not. The first andsecond nucleotide sequences of a blocked nucleic acid molecule may be onthe same nucleic acid molecule (e.g., for single-strand embodiments) oron separate nucleic acid molecules (e.g., for double-strandembodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the secondnucleotide sequence converts the blocked nucleic acid molecule to asingle-strand unblocked nucleic acid molecule. The unblocked nucleicacid molecule contains only the first nucleotide sequence, which hassufficient complementarity to hybridize to the gRNA of RNP2, therebyactivating the trans-cleavage activity of RNP2.

In some embodiments, the second nucleotide sequence at least partiallyhybridizes to the first nucleotide sequence, resulting in a secondarystructure containing at least one loop (e.g., hairpin loops, tetraloops,pseudoknots, junctions, kissing hairpins, internal loops, bulges, andmultibranch loops). Such loops block the nucleic acid molecule frombinding or incorporating into an RNP complex thereby initiating cis- ortrans-cleavage (see, e.g., the exemplary structures in FIGS. 2C-2F).

In some embodiments, the blocked nucleic acid molecule may contain aprotospacer adjacent motif (PAM) sequence, or partial PAM sequence,positioned between the first and second nucleotide sequences, where thefirst sequence is 5′ to the PAM sequence, or partial PAM sequence, (seeFIG. 2G). Inclusion of a PAM sequence may increase the reaction kineticsinternalizing the unblocked nucleic acid molecule into RNP2 and thusdecrease the time to detection. In other embodiments, the blockednucleic acid molecule does not contain a PAM sequence.

In some embodiments, the blocked nucleic acid molecules (i.e., highK_(d) nucleic acid molecules in relation to binding to RNP2) of thedisclosure may include a structure represented by Formula I (e.g., FIG.2C), Formula II (e.g., FIG. 2D), Formula III (e.g., FIG. 2E), or FormulaIV (e.g., FIG. 2F) wherein Formulas I-IV are in the 5′-to-3′ direction:

A-(B-L)J-C-M-T-D (Formula I);

-   -   wherein A is 0-15 nucleotides in length;    -   B is 4-12 nucleotides in length;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10;    -   C is 4-15 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then A-(B-L)J-C and T-D are separate nucleic acid        strands;    -   T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or        17-25) and comprises a sequence complementary to B and C; and    -   D is 0-10 nucleotides in length and comprises a sequence        complementary to A;

D-T-T′-C-(L-B)J-A (Formula II);

-   -   wherein D is 0-10 nucleotides in length;    -   T-T′ is 17-135 nucleotides in length (e.g., 17-100, 17-50, or        17-25);    -   T′ is 1-10 nucleotides in length and does not hybridize with T;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T;    -   L is 3-25 nucleotides in length and does not hybridize with T;    -   B is 4-12 nucleotides in length and comprises a sequence        complementary to T;    -   J is an integer between 1 and 10;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D;

T-D-M-A-(B-L)J-C (Formula III);

-   -   wherein T is 17-135 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-10 nucleotides in length;    -   M is 1-25 nucleotides in length or is absent, wherein if M is        absent then T-D and A-(B-L)J-C are separate nucleic acid        strands;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D;    -   B is 4-12 nucleotides in length and comprises a sequence        complementary to T;    -   L is 3-25 nucleotides in length;    -   J is an integer between 1 and 10; and    -   C is 4-15 nucleotides in length;

T-D-M-A-Lp-C (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,        or 17-25);    -   D is 0-15 nucleotides in length;    -   M is 1-25 nucleotides in length;    -   A is 0-15 nucleotides in length and comprises a sequence        complementary to D; and    -   L is 3-25 nucleotides in length;    -   p is 0 or 1;    -   C is 4-15 nucleotides in length and comprises a sequence        complementary to T.

-   In alternative embodiments of any of these molecules, T (or T-T′)    can have a maximum length of 1000 nucleotides, e.g., at most 750, at    most 500, at most 400, at more 300, at most 250, at most 200, at    most 150, at most 135, at most 100, at most 75, at most 50, or at    most 25 nucleotides.

Nucleotide mismatches can be introduced in any of the above structurescontaining double-strand segments (for example, where M is absent inFormula I or Formula III) to reduce the melting temperature (T_(m)) ofthe segment such that once the loop (L) is cleaved, the double-strandsegment is unstable and dehybridizes rapidly. The percentage ofnucleotide mismatches of a given segment may vary between 0% and 50%;however, the maximum number of nucleotide mismatches is limited to anumber where the secondary loop structure still forms. “Segments” in theabove statement refers to A, B, and C. In other words, the number ofhybridized bases can be less than or equal to the length of eachdouble-strand segment and vary based on number of mismatches introduced.

In any blocked nucleic acid molecule having the structure of Formula I,III, or IV, T will have sequence complementarity to a nucleotidesequence (e.g., a spacer sequence) within a gRNA of RNP2. The nucleotidesequence of T is to be designed such that hybridization of T to the gRNAof RNP2 activates the trans-nuclease activity of RNP2. In any blockednucleic acid molecule having structure of Formula II, T-T′ will havesequence complementarity to a sequence (e.g., a spacer sequence) withinthe gRNA of RNP2. The nucleotide sequence of T-T′ is to be designed suchthat hybridization of T-T′ to the gRNA of RNP2 activates thetrans-nuclease activity of RNP2. For T or T-T′, full complementarity tothe gRNA is not necessarily required, provided there is sufficientcomplementarity to cause hybridization and trans-cleavage activation ofRNP2.

In any of the foregoing embodiments, the blocked nucleic acid moleculesof the disclosure may and preferably do further contain a reportermoiety attached thereto such that cleavage of the blocked nucleic acidreleases a signal from the reporter moiety. (See FIG. 4 , mechanismsdepicted at center and bottom.)

Also, in any of the foregoing embodiments, the blocked nucleic acidmolecule may be a modified or non-naturally occurring nucleic acidmolecule. In some embodiments, the blocked nucleic acid molecules of thedisclosure may further contain a locked nucleic acid (LNA), a bridgednucleic acid (BNA), and/or a peptide nucleic acid (PNA). The blockednucleic acid molecule may contain a modified or non-naturally occurringnucleoside, nucleotide, and/or internucleoside linkage, such as a2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modifiednucleoside, and a phosphorothioate (PS) bond, any other nucleic acidmolecule modifications described above, and any combination thereof.

FIG. 2G at left shows an exemplary single-strand blocked nucleic acidmolecule and how the configuration of this blocked nucleic acid moleculeis able to prevent (or significantly prevent) undesired unwinding of theblocked nucleic acid molecule (or blocked primer molecule) and R-loopformation with an RNP complex, thereby blocking activation of thetrans-cleavage activity of RNP2. The single-strand blocked nucleic acidmolecule is self-hybridized and comprises: a target strand (TS) sequencecomplementary to the gRNA (e.g., crRNA) of RNP2; a cleavable non-targetstrand (NTS) sequence that is partially hybridized (e.g., it containssecondary loop structures) to the TS sequence; and a protospaceradjacent motif (PAM) sequence (e.g., 5′ NAAA 3′) that is specificallylocated at the 3′ end of the TS sequence. An RNP complex with 3′→5′diffusion (e.g., 1D diffusion) initiates R-loop formation upon PAMrecognition. R-loop formation is completed upon a stabilizing >17 basehybridization of the TS to the gRNA of RNP2; however, because of theorientation of the PAM sequence relative to the secondary loopstructure(s), the blocked nucleic acid molecule sterically prevents thetarget strand from hybridizing with the gRNA of RNP2, thereby blockingthe stable R-loop formation required for the cascade reaction.

FIG. 2G at right shows the blocked nucleic acid molecule being unblockedvia trans-cleavage (e.g., by RNP1) and subsequent dehybridization of thenon-target strand's secondary loop structures, followed by binding ofthe target strand to the gRNA of RNP2, thereby completing stable R-loopformation and activating the trans-cleavage activity of the RNP2complex.

In some embodiments, the blocked nucleic acid molecules provided hereinare circular DNAs, RNAs or chimeric (DNA-RNA) molecules (FIG. 2H), andthe blocked nucleic acid molecules may include different basecompositions depending on the Cas enzyme used for RNP1 and RNP2. For thecircular design of blocked nucleic acid molecules, the 5′ and 3′ endsare covalently linked together. This configuration makes internalizationof the blocked nucleic acid molecule into RNP2—and subsequent RNP2activation—sterically unfavorable, thereby blocking the progression ofthe cascade assay. Thus, RNP2 activation (e.g., trans-cleavage activity)happens after cleavage of a portion of the blocked nucleic acid moleculefollowed by linearization and internalization of unblocked nucleic acidmolecule into RNP2.

In some embodiments, the blocked nucleic acid molecules aretopologically circular molecules with 5′ and 3′ portions hybridized toeach other using DNA, RNA, LNA, BNA, or PNA bases which have a very highmelting temperature (Tm). The high Tm causes the structure toeffectively behave as a circular molecule even though the 5′ and 3′ endsare not covalently linked. The 5′ and 3′ ends can also have basenon-naturally occurring modifications such as phosphorothioate bonds toprovide increased stability.

In embodiments where the blocked nucleic acid molecules are circularized(e.g., circular or topologically circular), as illustrated in FIG. 2H,each blocked nucleic acid molecule includes a first region, which is atarget sequence specific to the gRNA of RNP2, and a second region, whichis a sequence that can be cleaved by nuclease enzymes of activated RNP1and/or RNP2. The first region may include a nuclease-resistant nucleicacid sequence such as, for example, a phosphorothioate group or othernon-naturally occurring nuclease-resistant base modifications, forprotection from trans-nucleic acid-guided nuclease activity. In someembodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas12a, thefirst region of the blocked nucleic acid molecule includes anuclease-resistant DNA sequence, and the second region of the blockednucleic acid molecule includes a cleavable DNA sequence. In otherembodiments, when the Cas enzyme in RNP1 is Cas12a and the Cas enzyme inRNP2 is Cas13a, the first region of the blocked nucleic acid moleculeincludes a nuclease-resistant RNA sequence, and the second region of theblocked nucleic acid molecule includes a cleavable DNA sequence and acleavable RNA sequence. In yet other embodiments, when the Cas enzyme inRNP1 is Cas13a and the Cas enzyme in RNP2 is Cas12a, the first region ofthe blocked nucleic acid molecule includes a nuclease-resistant DNAsequence, and the second region of the blocked nucleic acid moleculeincludes a cleavable DNA sequence and a cleavable RNA sequence. In someother embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas13a,the first region of the blocked nucleic acid molecule includes anuclease-resistant RNA sequence, and the second region of the blockednucleic acid molecule includes a cleavable RNA sequence.

The Signal Boosting Cascade Assay Employing Blocked Primer Molecules

The blocked nucleic acid molecules described above may also be blockedprimer molecules. Blocked primer molecules include a sequencecomplementary to a primer binding domain (PBD) on a template molecule(see description below in reference to FIGS. 3A and 3B) and can have thesame general structures as the blocked nucleic acid molecules describedabove. A PBD serves as a nucleotide sequence for primer hybridizationfollowed by primer polymerization by a polymerase. In any of Formulas I,II, or III described above, the blocked primer nucleic acid molecule mayinclude a sequence complementary to the PBD on the 5′ end of T. Theunblocked primer nucleic acid molecule can bind to a template moleculeat the PBD and copy the template molecule via polymerization by apolymerase.

Specific embodiments of the cascade assay which utilize blocked primermolecules and are depicted in FIGS. 3A and 3B. In the embodiments usingblocked nucleic acid molecules described above, activation of RNP1 bybinding of N nucleotides of the target nucleic acid molecules orcis-cleavage of the target nucleic acid molecules initiatestrans-cleavage of the blocked nucleic acid molecules which were used toactivate RNP2—that is, the unblocked nucleic acid molecules are a targetsequence for the gRNA in RNP2. In contrast, in the embodiments usingblocked primers activation of RNP1 and trans-cleavage unblocks a blockedprimer molecule that is then used to prime a template molecule forextension by a polymerase, thereby synthesizing synthesized activatingmolecules that are the target sequence for the gRNA in RNP2.

FIG. 3A is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and lineartemplate molecules. At left of FIG. 3A is a cascade assay reaction mixcomprising 1) RNP 1 s (301) (only one RNP1 is shown); 2) RNP2s (302); 3)linear template molecules (330) (which is the non-target strand); 4) acircular blocked primer molecule (334) (i.e., a high K_(d) molecule);and 5) a polymerase (338), such as a 129 polymerase. The linear templatemolecule (330) (non-target strand) comprises a PAM sequence (331), aprimer binding domain (PBD) (332) and, optionally, a nucleosidemodification (333) to protect the linear template molecule (330) from3′→5′ exonuclease activity. Blocked primer molecule (334) comprises acleavable region (335) and a complement to the PBD (332) on the lineartemplate molecule (330).

Upon addition of a sample comprising a target nucleic acid of interest(304) (capable of complexing with the gRNA in RNP1 (301)), the targetnucleic acid of interest (304) is bound by with and activates RNP1 (305)but does not interact with or activate RNP2 (302). Once activated, RNP1cuts the target nucleic acid of interest (304) via sequence specificcis-cleavage, which activates non-specific trans-cleavage of othernucleic acids present in the reaction mix, including at least one of theblocked primer molecules (334). The circular blocked primer molecule(334) (i.e., a high K_(d) molecule, where high K_(d) relates to bindingto RNP2) upon cleavage becomes an unblocked linear primer molecule (344)(a low K_(d) molecule, where low K_(d) relates to binding to RNP2),which has a region (336) complementary to the PBD (332) on the lineartemplate molecule (330) and can bind to the linear template molecule(330).

Once the unblocked linear primer molecule (344) and the linear templatemolecule (330) are hybridized (i.e., hybridized at the PBD (332) of thelinear template molecule (330) and the PBD complement (336) on theunblocked linear primer molecule (344)), 3′→5′ exonuclease activity ofthe polymerase (338) removes the unhybridized single-stranded DNA at theend of the unblocked primer molecule (344) and the polymerase (338) cancopy the linear template molecule (330) to produce a synthesizedactivating molecule (346) which is a complement of the non-targetstrand, which is the target strand. The synthesized activating molecule(346) is capable of activating RNP2 (302→308). As described above,because the nucleic acid-guided nuclease in the RNP2 (308) complexexhibits (that is, possesses) both cis- and trans-cleavage activity,more blocked primer molecules (334) become unblocked primer molecules(344) triggering activation of more RNP2s (308) and more trans-cleavageactivity in a cascade. As stated above in relation to blocked andunblocked nucleic acid molecules (both linear and circular), theunblocked primer molecule has a higher binding affinity for the gRNA inRNP2 than does the blocked primer molecule, although there may be some“leakiness” where some blocked primer molecules are able to interactwith the gRNA in RNP2. However, an unblocked primer molecule has asubstantially higher likelihood than a blocked primer molecule tohybridize with the gRNA of RNP2.

FIG. 3A at bottom depicts the concurrent activation of reportermoieties. Intact reporter moieties (309) comprise a quencher (310) and afluorophore (311). As described above in relation to FIG. 1B, thereporter moieties are also subject to trans-cleavage by activated RNP1(305) and RNP2 (308). The intact reporter moieties (309) becomeactivated reporter moieties (312) when the quencher (310) is separatedfrom the fluorophore (311), and the fluorophore emits a fluorescentsignal (313). Signal strength increases rapidly as more blocked primermolecules (334) become unblocked primer molecules (344) generatingsynthesized activating molecules (346) and triggering activation of moreRNP2 (308) complexes and more trans-cleavage activity of the reportermoieties (309). Again, here the reporter moieties are shown as separatemolecules from the blocked nucleic acid molecules, but otherconfigurations may be employed and are discussed in relation to FIG. 4 .Also, as with the cascade assay embodiment utilizing blocked nucleicacid molecules that are not blocked primers, with the exception of thegRNA in RNP1, the cascade assay components stay the same no matter whattarget nucleic acid(s) of interest are being detected.

FIG. 3B is a diagram showing the sequence of steps in an exemplarycascade assay involving circular blocked primer molecules and circulartemplate molecules. The cascade assay of FIG. 3B differs from thatdepicted in FIG. 3A by the configuration of the template molecule. Wherethe template molecule in FIG. 3A was linear, in FIG. 3B the templatemolecule is circular. At left of FIG. 3B is a cascade assay reaction mixcomprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) acircular template molecule (352) (non-target strand); 4) a circularblocked primer molecule (334); and 5) a polymerase (338), such as a Φ29polymerase. The circular template molecule (352) (non-target strand)comprises a PAM sequence (331) and a primer binding domain (PBD) (332).Blocked primer molecule (334) comprises a cleavable region (335) and acomplement to the PBD (332) on the circular template molecule (352).

Upon addition of a sample comprising a target nucleic acid of interest(304) (capable of complexing with the gRNA in RNP1 (301)), the targetnucleic acid of interest (304) binds to and activates RNP1 (305) butdoes not interact with or activate RNP2 (302). Once activated, RNP1 cutsthe target nucleic acid of interest (304) via sequence specificcis-cleavage, which activates non-specific trans-cleavage of othernucleic acids present in the reaction mix, including at least one of theblocked primer molecules (334). The circular blocked primer molecule(334), upon cleavage, becomes an unblocked linear primer molecule (344),which has a region (336) complementary to the PBD (332) on the circulartemplate molecule (352) and can hybridize with the circular templatemolecule (352).

Once the unblocked linear primer molecule (344) and the circulartemplate molecule (352) are hybridized (i.e., hybridized at the PBD(332) of the circular template molecule (352) and the PBD complement(336) on the unblocked linear primer molecule (344)), 3′→5′ exonucleaseactivity of the polymerase (338) removes the unhybridizedsingle-stranded DNA at the 3′ end of the unblocked primer molecule(344). The polymerase (338) can now use the circular template molecule(352) (non-target strand) to produce concatenated activating nucleicacid molecules (360) (which are concatenated target strands), which willbe cleaved by the trans-cleavage activity of activated RNP1. The cleavedregions of the concatenated synthesized activating molecules (360)(target strand) are capable of activating the RNP2 (302→308) complex.

As described above, because the nucleic acid-guided nuclease in RNP2(308) comprises both cis- and trans-cleavage activity, more blockedprimer molecules (334) become unblocked primer molecules (344)triggering activation of more RNP2s (308) and more trans-cleavageactivity in a cascade. FIG. 3B at bottom depicts the concurrentactivation of reporter moieties. Intact reporter moieties (309) comprisea quencher (310) and a fluorophore (311). As described above in relationto FIG. 1B, the reporter moieties are also subject to trans-cleavage byactivated RNP1 (305) and RNP2 (308). The intact reporter moieties (309)become activated reporter moieties (312) when the quencher (310) isseparated from the fluorophore (311), and the fluorescent signal (313)is unquenched and can be detected. Signal strength increases rapidly asmore blocked primer molecules (334) become unblocked primer molecules(344) generating synthesized activating nucleic acid molecules andtriggering activation of more RNP2s (308) and more trans-cleavageactivity of the reporter moieties (309). Again, here the reportermoieties are shown as separate molecules from the blocked nucleic acidmolecules, but other configurations may be employed and are discussed inrelation to FIG. 4 . Also note that as with the other embodiments of thecascade assay, in this embodiment, with the exception of the gRNA inRNP1, the cascade assay components stay the same no matter what targetnucleic acid(s) of interest are being detected.

The polymerases used in the “blocked primer molecule” embodiments serveto polymerize a reverse complement strand of the template molecule(non-target strand) to generate a synthesized activating molecule(target strand) as described above. In some embodiments, the polymeraseis a DNA polymerase, such as a BST, T4, or Therminator polymerase (NewEngland BioLabs Inc., Ipswich Mass., USA). In some embodiments, thepolymerase is a Klenow fragment of a DNA polymerase. In some embodimentsthe polymerase is a DNA polymerase with 5′→3′ DNA polymerase activityand 3′→5′ exonuclease activity, such as a Type I, Type II, or Type IIIDNA polymerase. In some embodiments, the DNA polymerase, including thePhi29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent®DNA polymerases (New England BioLabs Inc., Ipswich Mass., USA), or anyactive portion or variant thereof. Also, a 3′ to 5′ exonuclease can beseparately used if the polymerase lacks this activity.

FIG. 4 depicts three mechanisms in which a cascade assay reaction canrelease a signal from a reporter moiety. FIG. 4 at top shows themechanism discussed in relation to FIGS. 2A, 3A and 3B. In thisembodiment, a reporter moiety 409 is a separate molecule from theblocked nucleic acid molecules present in the reaction mix. Reportermoiety (409) comprises a quencher (410) and a fluorophore (411). Anactivated reporter moiety (412) emits a signal from the fluorophore(411) once it has been physically separated from the quencher (410).

Reporter Moiety Configurations

FIG. 4 at center shows a blocked nucleic acid molecule (403), which isalso a reporter moiety. In addition to quencher (410) and fluorophore(411), a blocking moiety (407) can be seen (see also blocked nucleicacid molecules 203 in FIG. 2A). Blocked nucleic acid molecule/reportermoiety (403) comprises a quencher (410) and a fluorophore (411). In thisembodiment of the cascade assay, when the blocked nucleic acid molecule(403) is unblocked due to trans-cleavage initiated by the target nucleicacid of interest binding to RNP1, the unblocked nucleic acid molecule(406) also becomes an activated reporter moiety with fluorophore (411)separated from quencher (410). Note both the blocking moiety (407) andthe quencher (410) are removed. In this embodiment, reporter signal isdirectly generated as the blocked nucleic acid molecules becomeunblocked. Embodiments of this schema can be used to supply the bulkymodifications to the blocked nucleic acid molecules described below.

FIG. 4 at the bottom shows that cis-cleavage of an unblocked nucleicacid molecule or a synthesized activating molecule at a PAM distalsequence by RNP2 generates a signal. Shown are activated RNP2 (408),unblocked nucleic acid molecule (461), quencher (410), and fluorophore(411) forming an activated RNP2 with the unblocked nucleic acid/reportermoiety intact (460). Cis-cleavage of the unblocked nucleic acid/reportermoiety (461) results in an activated RNP2 with the reporter moietyactivated (462), comprising the activated RNP2 (408), the unblockednucleic acid molecule with the reporter moiety activated (463), quencher(410) and fluorophore (411). Embodiments of this schema also can be usedto supply the bulky modifications to the blocked nucleic acid moleculesdescribed below, and in fact a combination of the configurations ofreporter moieties shown in FIG. 4 at center and at bottom may be used.

Preventing Undesired Blocked Nucleic Acid Molecule Unwinding

The present disclosure improves upon the signal cascade assay describedin U.S. Ser. Nos. 17/861,207; 17/861,208; and 17/861,209 by addressingthe problem with undesired “unwinding” of the blocked nucleic acidmolecule. As described above in detail in relation to FIGS. 1B, 2A, 2B,2G, 3A, 3B, and 4 , the cascade assay is initiated when a target nucleicacid of interest binds to and activates a first pre-assembledribonucleoprotein complex (RNP1). The gRNA of RNP1 (gRNA1), comprising asequence complementary to the target nucleic acid of interest, guidesRNP1 to the target nucleic acid of interest. Upon binding of the targetnucleic acid of interest to RNP1, RNP1 becomes activated, and the targetnucleic acid of interest is cleaved in a sequence specific manner (i.e.,cis-cleavage) while also triggering non-sequence specific,indiscriminate trans-cleavage activity which unblocks the blockednucleic acid molecules in the reaction mix. The unblocked nucleic acidmolecules can then activate a second pre-assembled ribonucleoproteincomplex (RNP2), where RNP2 comprises a second gRNA (gRNA2) comprising asequence complementary to the unblocked nucleic acid molecules, and atleast one of the unblocked nucleic acid molecules is cis-cleaved in asequence specific manner. Binding of the unblocked nucleic acid moleculeto RNP2 leads to cis-cleavage of the unblocked nucleic acid molecule andnon-sequence specific, indiscriminate trans-cleavage activity by RNP2,which in turn unblocks more blocked nucleic acid molecules (and reportermoieties) in the reaction mix activating more RNP2s. Each newlyactivated RNP2 activates more RNP2s, which in turn cleave more blockednucleic acid molecules and reporter moieties in a reaction cascade,where all or most of the signal generated comes from the trans-cleavageactivity of RNP2.

The improvement to the signal boost cascade assay described herein isdrawn to preventing undesired unwinding of the blocked nucleic acidmolecules in the reaction mix before the blocked nucleic acid moleculesare unblocked via trans-cleavage; that is, preventing undesiredunwinding that happens not as a result of unblocking due totrans-cleavage subsequent to cis-cleavage of the target nucleic acid ofinterest or trans-cleavage of unblocked nucleic acid molecules, but dueto other factors. For a description of undesired unwinding, please seeFIG. 1C and the attendant description herein. Minimizing undesiredunwinding serves two purposes. First, preventing undesired unwindingthat happens not as a result of designed or engineered unblocking leadsto a “leaky” cascade assay system, which in turn leads to non-specificsignal generation and false positives.

Second, preventing undesired unwinding limits non-specific interactionsbetween the nucleic acid-guided nucleases (here, the RNP2s) and blockednucleic acid molecules (i.e., the target nucleic acids for RNP2) suchthat only blocked nucleic acid molecules that become unblocked due totrans-cleavage activity react with the nucleic acid-guided nucleases.This “fidelity” in the cascade assay leads primarily to desiredinteractions and limits “wasteful” interactions where the nucleicacid-guided nucleases are essentially interacting with blocked nucleicacid molecules rather than interacting with unblocked nucleic acidmolecules. That is, if unwinding is minimized the nucleic acid-guidednucleases are focused on desired interactions which then leads toimmediate signal generation in the cascade assay. Preventing undesiredunwinding leads to a more efficient cascade assay system providing moreaccurate quantification yet with the rapid results characteristic of thecascade assay (see FIGS. 10A-10H and 12 below).

Ratio of RNP2 to Blocked Nucleic Acid Molecules or Blocked Primers

In one modality to prevent undesired unwinding, the present disclosuredescribes using an unconventional ratio of blocked nucleic acid molecule(i.e., the target molecule for RNP2) and an RNP complex, here RNP2. Theunconventional ratio may be used along with the blocked nucleic acidmolecules and RNP2s described above as a primary method for minimizingunwinding or may be used in combination with the other modalitiesdescribed below to minimize unwinding even more. For example, if onewere to design an ideal blocked nucleic acid molecule having an“infinite K_(d)” such as, e.g., through design of the blocked nucleicacid molecule (or blocked primer molecule) and/or inclusion of bulkymodifications on the blocked nucleic acid molecule (or blocked primermolecule), the ratio of blocked nucleic acid molecules to RNP2s wouldnot affect the reaction mix to any discernable degree. The common wisdomof the ratio of enzyme to target (here, RNP2 to blocked nucleic acidmolecule) is that results are achieved—a signal is generated—when thereis a high concentration of nucleic acid-guided nuclease (i.e., RNPcomplex) and a lower concentration of target or, stated another way,when there is a significant excess of nucleic acid-guided nuclease totarget. As described above, in CRISPR detection/diagnostic assayprotocols known to date, the CRISPR enzyme (i.e., nucleic acid-guidednuclease) is far in excess of blocked nucleic acid molecules (see, Sun,et al., J. of Translational Medicine, 12:74 (2021); Broughton, et al.,Nat. Biotech., 38:870-74 (2020); and Lee, et al., PNAS, 117(41):25722-31(2020)). However, in a cascade assay system where the nucleicacid-guided nuclease (or RNP complex) is in excess of the targets (here,the blocked nucleic acid molecules), the nucleic acid-guided nucleasesencounter the blocked nucleic acid molecules repeatedly, probing theblocked nucleic acid molecules and subjecting them to unwinding. If theblocked nucleic acid molecules are probed and unwound repeatedly, theyfinally unwind which then triggers activation of RNP2 and cis-cleavageof the blocked nucleic acid molecule even in the absence of a targetnucleic acid of interest and the trans-cleavage activity generatedthereby.

However, by adjusting the ratio of RNP2 to blocked nucleic acidmolecules such that there is an excess of blocked nucleic acid moleculesto RNP2, any one blocked nucleic acid molecule may be probed by RNP2;however, the likelihood that any one blocked nucleic acid molecule willbe probed repeatedly (and thus unwound) is much lower. If a blockednucleic acid molecule is probed but then has time to re-hybridize or“recover”, that blocked nucleic acid molecule will stay blocked, willnot be subject to non-specific unwinding, and will not triggeractivation of RNP2. That is, how often any one blocked nucleic acidmolecule is probed is important. As long as an improperly probed blockednucleic acid has time to re-hybridize after unwinding, there is far lesschance that the blocked nucleic acid will be unblocked (i.e., unwound)and will trigger signal generation. That is, preventing non-specificunwinding of the blocked nucleic acid molecules makes the nucleicacid-guided nuclease available for desired unwinding interactions.

In order to prevent non-specific unwinding as described herein, theratio of blocked nucleic acid molecules to RNP2 should be about 50:1, orabout 40:1, or about 35:1, or about 30:1, or about 25:1, or about 20:1,or about 15:1, or about 10:1, or about 7.5:1, or about 5:1, or about4:1, or about 3:1, or about 2.5:1, or about 2:1, or about 1.5:1, or atleast where the molar concentration of blocked nucleic acid molecules isequal to or greater than the molar concentration of RNP2s. As notedabove, the signal amplification cascade assay reaction mixture typicallycontains about 1 fM to about 1 mM of a given RNP2, or about 1 pM toabout 500 μM of a given RNP2, or about 10 pM to about 100 μM of a givenRNP2; thus, the signal amplification cascade assay reaction mixturetypically contains about 2.5 fM to about 2.5 mM blocked nucleic acidmolecules, or about 2.5 pM to about 1.25 mM blocked nucleic acidmolecules, or about 25 pM to about 250 μM blocked nucleic acidmolecules. That is, the reaction mixture contains about 6×10⁴ to about6×10¹⁴ RNP2s per microliter (μl) or about 6×10⁶ to about 6×10¹² RNP2sper microliter (μl) and thus about 6×10⁴ to about 6×10¹⁴ RNP2s permicroliter (μl) or about 6×10⁶ to about 6×10¹² blocked nucleic acidmolecules per microliter (μl). Note, the ratios may be used along withthe blocked nucleic acid molecules and RNP2s described above as aprimary method for minimizing unwinding or the ratios of blocked nucleicacid molecules to RNP2s may be used in combination with the othermodalities described below to further minimize unwinding. Again, if onewere to design an ideal blocked nucleic acid molecule having an“infinite K_(d)”, the ratio of blocked nucleic acid molecules to RNP2swould not affect the reaction mix to any discernable degree and theratios of blocked nucleic acid molecules to RNP2s would not necessarilybe within these ranges.

Variant Engineered Nucleic Acid-Guided Nucleases

In some embodiments, the protein sequence of the Cas12a nucleicacid-guided nuclease is modified, with e.g., mutations to the domainsthat interact with the PAM region or surrounding sequences on theblocked nucleic acid molecules (see Shin et al., Front. Genet., 11:1577(2021); doi: 10.3389/fgene.2020.571591, herein incorporated byreference; and Yamano et al., Mol. Cell, 67(4): 633-645 (2017); doi:10.1016/j.molcel.2017.06.035, herein incorporated by reference) suchthat the variant engineered nucleic acid-guided nuclease has reduced (orabsent) PAM specificity, relative to the unmodified or wildtype nucleicacid-guided nuclease and reduced cleavage activity in relation to doublestrand DNA with or without a PAM. Such enzymes are referred to herein assingle-strand-specific Cas12a nucleic acid-guided nucleases or variantengineered nucleic acid-guided nucleases.

FIG. 5 is a simplified block diagram of an exemplary method 500 fordesigning, synthesizing and screening variant nucleic acid-guidednucleases. In a first step, mutations or modifications to a nucleicacid-guided nuclease are designed 502, based on, e.g., homology torelated nucleic acid-guided nucleases, predicted protein structure andactive site configuration, and mutagenesis modeling. For assessment ofhomologies to other nucleic acid-guided nucleases, amino acid sequencesmay be found in publicly available databases known to those with skillin the art, including, e.g., Protein DataBank Europe (PDBe), ProteinDatabank Japan (PDBj), SWISS-PROT, GenBank, RefSeq, TrEMBL, PROSITE,DisProt, InterPro, PIR-International, and PRF/SEQDB. Amino acid homologyalignments for purposes of determining similarities to known nucleicacid-guided nucleases can be performed using CUSTALW, CUSTAL OMEGA,COBALT: Multiple Alignment Tool; SIM; and PROBCONS.

For protein engineering and amino acid substitution model predictionsfor each of the desired mutations, protein modeling software such asSWISS-MODEL, HHpred, I-TASSER, IntFOLD, RaptorX, FoldX, Rosetta, andtrRosetta may be used to simulate the structural change(s) and tocalculate various parameters due to the structural changes as a resultof the amino acid substitution(s), including root mean square deviation(RMSD) value in Angstrom units (i.e., a measurement of the differencebetween the backbones of the initial nucleic acid-guided nuclease andthe mutated nucleic acid nucleic acid-guided nuclease) and changes tothe number of hydrogen bonds and conformation in the active site. Forthe methods used to generate the variant engineered nucleic acid-guidednucleases described herein, see Example VII below.

Following modelling, coding sequences for the variant nucleicacid-guided nucleases that appear to deliver desired properties aresynthesized and inserted into an expression vector 504. Methods forsite-directed mutagenesis are known in the art, including PCR-basedmethods such as traditional PCR, where primers are designed to includethe desired change; primer extension, involving incorporating mutagenicprimers in independent nested PCR before combining them in the finalproduct; and inverse PCR. Additionally, CRISPR gene editing may beperformed to introduce the desired mutation or modification to thenucleic acid-guided nuclease coding sequence. The mutated (variant)coding sequences are inserted into an expression vector backbonecomprising regulatory sequences such as enhancer and promoter regions.The type of expression vector (e.g., plasmid or viral vector) will varydepending on the type of cells to be transformed.

At step 506, cells of choice are transformed with the variant expressionvectors. A variety of delivery systems may be used to introduce (e.g.,transform or transfect) the expression vectors into a host cell,including the use of yeast systems, lipofection systems, microinjectionsystems, biolistic systems, virosomes, liposomes, immunoliposomes,polycations, lipid:nucleic acid conjugates, virions, artificial virions,viral vectors, electroporation, cell permeable peptides, nanoparticles,nanowires, exosomes. Once cells are transformed (or transfected), thetransformants are allowed to recover and grow.

Following transformation, the cells are screened for expression ofnucleic acid-guided nucleases with desired properties 508, such as cutactivity or lack thereof, paste activity or lack thereof, PAMrecognition or changes thereto, stability and the ability to form RNPsat various temperatures, and/or cis- and trans-cleavage activity atvarious temperatures. The assays used to screen the variant nucleicacid-guided nucleases will vary depending on the desired properties, butmay include in vitro and in vivo PAM depletion, assays for editingefficiency such as a GFP to BFP assay, and, as used to assess thevariant nucleic acid-guided nucleases described herein, in vitrotranscription/translation (IVTT) assays were used to measure in vitrotrans cleavage with both dsDNA and ssDNA and with and without thepresence of a PAM in the blocked nucleic acid molecules, where dsDNAshould not activate trans-cleavage regardless of the presence of PAMsequence.

After screening the variant nucleic acid-guided nucleases via the IVTTassays, variants with the preferred properties are identified andselected 510. At this point, a variant may be chosen 512 to go forwardinto production for use in, e.g., the CRISPR cascade systems describedherein; alternatively, promising mutations and/or modifications may becombined 514 and the construction, screening and identifying process isrepeated.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease may not recognize one or more of the following PAMor partial PAM sequences (listed from 5′ to 3′): TTTN, TTTV, CTTA, CTTV,TCTV, TTCV, YTV, or YTN wherein “A” represents adenine, “C” representscytosine, “T” represents thymine, “G” represents guanine, “V” representsguanine or cytosine or adenine, “Y” represents guanine or adenine, and“N” represents any nucleotide. In some embodiments, the Cas12a nucleicacid-guided nuclease may have reduced recognition for one or more of thefollowing PAM or partial PAM sequences (listed from 5′ to 3′): TTTN,TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN. The single-strand-specificCas12a nucleic acid-guided nucleases described herein may have at least50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or 100%, such as about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 100%) reduced recognition (i.e., specificity) forone or more of the following PAM or partial PAM sequences (listed from5′ to 3′) : TTTN, TTTV, CTTA, CTTV, TCTV, TTCV, YTV, or YTN.

Exemplary wild type (WT) Cas12a protein sequences are described in Table7 below. FIG. 6A shows the result of protein structure prediction usingRosetta and SWISS modeling of wildtype LbCas12a (Lachnospriaceaebacterium Cas12a), and FIG. 6B shows the result of example mutations onthe LbCas12a protein structure prediction using Rosetta and SWISSmodeling of LbCas12a and indicating the PAM regions (described in moredetail in relation to Example VII). Any of these sequences (e.g., SEQ IDNOs: 1-15 and homologs or orthologs thereof) may be modified, asdescribed herein, to generate a single-strand-specific nucleicacid-guided nuclease.

TABLE 7 Exemplary wild type Cas12a nucleic acid-guided nucleases SpeciesSEQ Name ID Reference ID NO: Protein Sequence Lachnospiraceae SEQMSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAED bacterium Cas12a IDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENK (LbCas12a) NO: 1ELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIAL PDD: 6KL9_AVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH Acidaminococcus SEQMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH sp. Cas12a IDYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETR (AsCas12a) NO: 2NALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFN NCBI Ref.:GKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDI WP_021736722.1STAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN QDWLAYIQELRN Candidatus SEQMNNYDEFTKLYPIQKTIRFELKPQGRTMEHLETFNFFEEDRDRAEK Methanoplasma IDYKILKEAIDEYHKKFIDEHLTNMSLDWNSLKQISEKYYKSREEKDK termitum NO: 3KVFLSEQKRMRQEIVSEFKKDDRFKDLFSKKLFSELLKEEIYKKGN (CtCas12a)HQEIDALKSFDKFSGYFIGLHENRKNMYSDGDEITAISNRIVNENFP NCBI Gene ID:KFLDNLQKYQEARKKYPEWIIKAESALVAHNIKMDEVFSLEYFNK 24818655VLNQEGIQRYNLALGGYVTKSGEKMMGLNDALNLAHQSEKSSKGRIHMTPLFKQILSEKESFSYIPDVFTEDSQLLPSIGGFFAQIENDKDGNIFDRALELISSYAEYDTERIYIRQADINRVSNVIFGEWGTLGGLMREYKADSINDINLERTCKKVDKWLDSKEFALSDVLEAIKRTGNNDAFNEYISKMRTAREKIDAARKEMKFISEKISGDEESIHIIKTLLDSVQQFLHFFNLFKARQDIPLDGAFYAEFDEVHSKLFAIVPLYNKVRNYLTKNNLNTKKIKLNFKNPTLANGWDQNKVYDYASLIFLRDGNYYLGIINPKRKKNIKFEQGSGNGPFYRKMVYKQIPGPNKNLPRVFLTSTKGKKEYKPSKEIIEGYEADKHIRGDKFDLDFCHKLIDFFKESIEKHKDWSKFNFYFSPTESYGDISEFYLDVEKQGYRMHFENISAETIDEYVEKGDLFLFQIYNKDFVKAATGKKDMHTIYWNAAFSPENLQDVVVKLNGEAELFYRDKSDIKEIVHREGEILVNRTYNGRTPVPDKIHKKLTDYHNGRTKDLGEAKEYLDKVRYFKAHYDITKDRRYLNDKIYFHVPLTLNFKANGKKNLNKMVIEKFLSDEKAHIIGIDRGERNLLYYSIIDRSGKIIDQQSLNVIDGFDYREKLNQREIEMKDARQSWNAIGKIKDLKEGYLSKAVHEITKMAIQYNAIVVMEELNYGFKRGRFKVEKQIYQKFENMLIDKMNYLVFKDAPDESPGGVLNAYQLTNPLESFAKLGKQTGILFYVPAAYTSKIDPTTGFVNLFNTSSKTNAQERKEFLQKFESISYSAKDGGIFAFAFDYRKFGTSKTDHKNVWTAYTNGERMRYIKEKKRNELFDPSKEIKEALTSSGIKYDGGQNILPDILRSNNNGLIYTMYSSFIAAIQMRVYDGKEDYIISPIKNSKGEFFRTDPKRRELPIDADANGAYNIALRGELTMRAIAEKFDPDSEKMAKLELKHKDWFEFMQTRGD Eubacterium SEQMNGNRSIVYREFVGVIPVAKTLRNELRPVGHTQEHIIQNGLIQEDEL eligens IDRQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSSP (EeCas12a) NO: 4SKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLLKEILPDFI NCBI Gene ID:KNYNQYDVKDKAGKLETLALFNGFSTYFTDFFEKRKNVFTKEAVS 41356122TSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGDWELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTKNNYNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETEKGNIIGKLKDIVNKYDELDEKRIYISKDFYETLSCFMSGNWNLITGCVENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDEKERNEFKNSNAKQYIREISNIITDTETAHLEYDDHISLIESEEKADEMKKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVPLYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILIRDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGANKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLIDYFKNSIEKHAEWRKYEFKFSATDSYSDISEFYREVEMQGYRIDWTYISEADINKLDEEGKIYLFQIYNKDFAENSTGKENLHTMYFKNIFSEENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQLDNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRTAQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMVVKYIAQNDDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVEKEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLIVEYNAIIAMEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKEKSVDEPGGLLKGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLEDNEINYADGHDIRIDMEKMDEDKKSEFFAQLLSLYKLTVQMRNSYTEAEEQENGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYCIALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRYE Moraxella SEQMLFQDFTHLYPLSKTVRFELKPIGKTLEHIHAKNFLNQDETMADM bovoculi Cas12a IDYQKVKAILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKD (Mb3Cas12a) NO: 5DGLQKQLKDLQAVLRKEIVKPIGNGGKYKAGYDRLFGAKLFKDG GenBank:KELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMY AKG12737.1SDEDKHTAIAYRLIHENLPRFIDNLQILATIKQKHSALYDQIINELTASGLDVSLASHLDGYHKLLTQEGITAYNTLLGGISGEAGSRKIQGINELINSHHNQHCHKSERIAKLRPLHKQILSDGMGVSFLPSKFADDSEVCQAVNEFYRHYADVFAKVQSLFDGFDDYQKDGIYVEYKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHYTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSDKSPEIRQLKELLDNALNVAHFAKLLTTKTTLHNQDGNFYGEFGALYDELAKIATLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLALLDKAHKKVFDNAPNTGKSVYQKMIYKLLPGPNKMLPKVFFAKSNLDYYNPSAELLDKYAQGTHKKGDNFNLKDCHALIDFFKAGINKHPEWQHFGFKFSPTSSYQDLSDFYREVEPQGYQVKFVDINADYINELVEQGQLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLVNPIYKLNGEAEIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGTQMTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQISQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNADRGYFEFHIDYAKFNDKAKNSRQIWKICSHGDKRYVYDKTANQNKGATIGVNVNDELKSLFTRYHINDKQPNLVMDICQNNDKEFHKSLMYLLKTLLALRYSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNR Francisella SEQMSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDY novicida Cas12a IDKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNL (FnCas12a) NO: 6QKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLIL UniProtKB/Swiss-WLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRK Prot: A0Q7Q2.1NVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQ NRNN Francisella SEQMSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDY tularensis subsp. IDKKAKQIIDKYHQFFIEEILSSVCISEDLLQNYSDVYFKLKKSDDDNL novicida FTG NO: 7QKDFKSAKDTIKKQISKYINDSEKFKNLFNQNLIDAKKGQESDLIL Cas12aWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRK (FnoCas12a)NVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIK NCBI Gene ID:KDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFN 60806594TIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQVAPKNLDNPSKKEQDLIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILSNFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDLLQASAEEDVKAIKDLLDQTNNLLHRLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLASGWDKNKESANTAILFIKDDKYYLGIMDKKHNKIFSDKAIEENKGEGYKKIVYKQIADASKDIQNLMIIDGKTVCKKGRKDRNGVNRQLLSLKRKHLPENIYRIKETKSYLKNEARFSRKDLYDFIDYYKDRLDYYDFEFELKPSNEYSDFNDFTNHIGSQGYKLTFENISQDYINSLVNEGKLYLFQIYSKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKETIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDNFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKGLMLLDRIKNNQEGKKLNLVIK NEEYFEFVQNRNNFlavobacteriales SEQ MKNNNMLNFTNKYQLSKTLRFELKPIGKTKENIIAKNILKKDEERAbacterium ID ESYQLMKKTIDGFHKHFIELAMQEVQKTKLSELEEFAELYNKSAEE (FbCas12a)NO: 8 KKKDDKFDDKFKKVQEALRKEIVKGFNSEKVKYYYSNIDKKILFT NCBI Gene ID:ELLKNWIPNEKMITELSEWNAKTKEEKEHLVYLDKEFENFTTYFG MBE7442138.1GFHKNRENMYTDKEQSTAIAYRLIHENLPKFLDNINIYKKVKEIPVLREECKVLYKEIEEYLNVNSIDEVFELSYYNKTLTQKDIDVYNLIIGGRTLEEGKKKIQGLNEYINLYNQKQEKKNRIPKLKILYKQILSDRDSISWLPESFEDDNEKTASQKVLEAINLYYRDNLLCFQPKDKKDTENVLEETKKLLAGLSTSDLSKIYIRNDRAITDISQALFKDYGVIKDALKFQFIQSFTIGKNGLSKKQEEAIEKHLKQKYFSIAEIENALFTYQSETDALKELKENSHPVVDYFINHFKAKKKEETDKDFDLIANIDAKYSCIKGLLNTPYPKDKKLYQRSKGDNDIDNIKAFLDALMELLHFVKPLALSNDSTLEKDQNFYSHFEPYYEQLELLIPLYNKVRNFAAKKPYSTEKFKLNFDNATLLNGWDKNKETDNTSVILRKDGLYYLAIMPQDNKNVFKDSPDLKANENCFEKMDYKQMALPMGFGAFVRKCFGTASQLGWNCPESCKNEEDKIIIKEDEVKNNRAEIIDCYKDFLNIYEKDGFQYKEYGFDFKESNKYESLREFFIDVEQQGYKITFQNISENYINQLVEDGKLYLFQIYNKDFSPYSKGKPNMHTMYWKALFDSENLKDVVYKLNGQAEVFYRKKSIEQKNIVTHKANEPIDNKNPKAKKKQSTFEYDLIKDKRYTVDKFQFHVPITLNFKATGNDYINQDVLTYLKNNPEVNIIGLDRGERHLIYLTLINQKGEILLQESLNTIVNKKYDIETPYHTLLQNKEDERAKARENWGVIENIKELKEGYISQVVHKIAKLMVEYNAIVVMEDLNTGFKRGRFKVEKQVYQKLEKMLIDKLNYLVFKDKDPSEVGGLYHALQLTNKFENFSKIGKQSGFLFYVPAWNTSKIDPTTGFVNLFNTKYESVPKAQEFFKKFKSIKFNSAENYFEFAFDYNDFTTRAEGTKTDWIVCTYGDRIKTFRNPDKVNQWDNQEVNLTEQFEDFFGKNNLIYGDGNCIKNQIILHDKKEFFEGLLHLLKLTLQMRNSITNSEVDYLISPVKNNKGEFYDSRKANNTLPKDADANGAYHIAKKGLVLLNRLKENEVEEFEKSKKVKDGKSQWLPNKDWLDFVQRNVEDMVVV Lachnospira SEQMNGNRSIVYREFVGVTPVAKTLRNELRPVGHTQEHIIQNGLIQEDE eligens IDLRQEKSTELKNIMDDYYREYIDKSLSGVTDLDFTLLFELMNLVQSS (Lb4Cas12a) NO: 9PSKDNKKALEKEQSKMREQICTHLQSDSNYKNIFNAKLFKEILPDFI NCBI Gene ID:KNYNQYDVKDKAGKLETVALFNGFSTYFTDFFEKRKNVFTKEAV MBS6299380.1STSIAYRIVHENSLIFLANMTSYKKISEKALDEIEVIEKNNQDKMGDWELNQIFNPDFYNMVLIQSGIDFYNEICGVVNAHMNLYCQQTRNNYNLFKMRKLHKQILAYTSTSFEVPKMFEDDMSVYNAVNAFIDETEKGNIIVKLKDIVNKYDELDEKRIYISKDFYETLSCFISGNWNLITGCVENFYDENIHAKGKSKEEKVKKAVKEDKYKSINDVNDLVEKYIDEKERNEFKNSNAKQYIREISNIITDTETAHLEYDEHISLIESEEKADEMKKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVPLYNRVRNYVTQKPYNSKKIKLNFQSPTLANGWSQSKEFDNNAIILIRDNKYYLAIFNAKNKPDKKIIQGNSDKKNDNDYKKMVYNLLPGANKMLPKVFLSKKGIETFKPSDYIISGYNAHKHIKTSENFDISFCRDLIDYFKNSIEKHAEWRKYEFKFSATDSYNDISEFYREVEMQGYRIDWTYISEADINKLDEEGKIYLFQIYNKYFAENSTGKENLHTMYFKNIFSEENLKDIIIKLNGQAELFYRRASVKNPVKHKKDSVLVNKTYKNQLDNGDVVRIPIPDDIYNEIYKMYNGYIKESDLSEAAKEYLDKVEVRTAQKDIVKDYRYTVDKYFIHTPITINYKVTARNNVNDMAVKYIAQNDDIHVIGIDRGERNLIYISVIDSHGNIVKQKSYNILNNYDYKKKLVEKEKTREYARKNWKSIGNIKELKEGYISGVVHEIAMLMVEYNAIIAMEDLNYGFKRGRFKVERQVYQKFESMLINKLNYFASKGKSVDEPGGLLRGYQLTYVPDNIKNLGKQCGVIFYVPAAFTSKIDPSTGFISAFNFKSISTNASRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGKTQWTVYTNGERLQSEFNNARRTGKTKSINLTETIKLLLKDNKINYADGHDVRIDMEKMDEDKNSEFFAQLLSLYKLTVQMRNSYTEAEEQEKGISYDKIISPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYCIALKGLYEVLKIKSEWTEDGFDRNCLKLPHAEWLDFIQNKRY E Moraxella SEQMLFQDFTHLYPLSKTVRFELKPIGRTLEHIHAKNFLSQDETMADMY bovoculi IDQKVKVILDDYHRDFIADMMGEVKLTKLAEFYDVYLKFRKNPKDD (MbCas12a) NO:GLQKQLKDLQAVLRKESVKPIGSGGKYKTGYDRLFGAKLFKDGK NCBI Gene ID: 10ELGDLAKFVIAQEGESSPKLAHLAHFEKFSTYFTGFHDNRKNMYS WP_046697655.1DEDKHTAIAYRLIHENLPRFIDNLQILTTIKQKHSALYDQIINELTASGLDVSLASHLDGYHKLLTQEGITAYNRIIGEVNGYTNKHNQICHKSERIAKLRPLHKQILSDGMGVSFLPSKFADDSEMCQAVNEFYRHYTDVFAKVQSLFDGFDDHQKDGIYVEHKNLNELSKQAFGDFALLGRVLDGYYVDVVNPEFNERFAKAKTDNAKAKLTKEKDKFIKGVHSLASLEQAIEHHTARHDDESVQAGKLGQYFKHGLAGVDNPIQKIHNNHSTIKGFLERERPAGERALPKIKSGKNPEMTQLRQLKELLDNALNVAHFAKLLTTKTTLDNQDGNFYGEFGVLYDELAKIPTLYNKVRDYLSQKPFSTEKYKLNFGNPTLLNGWDLNKEKDNFGVILQKDGCYYLALLDKAHKKVFDNAPNTGKNVYQKMVYKLLPGPNKMLPKVFFAKSNLDYYNPSAELLDKYAKGTHKKGDNFNLKDCHALIDFFKAGINKHPEWQHFGFKFSPTSSYRDLSDFYREVEPQGYQVKFVDINADYIDELVEQGKLYLFQIYNKDFSPKAHGKPNLHTLYFKALFSEDNLADPIYKLNGEAQIFYRKASLDMNETTIHRAGEVLENKNPDNPKKRQFVYDIIKDKRYTQDKFMLHVPITMNFGVQGMTIKEFNKKVNQSIQQYDEVNVIGIDRGERHLLYLTVINSKGEILEQRSLNDITTASANGTQVTTPYHKILDKREIERLNARVGWGEIETIKELKSGYLSHVVHQINQLMLKYNAIVVLEDLNFGFKRGRFKVEKQIYQNFENALIKKLNHLVLKDKADDEIGSYKNALQLTNNFTDLKSIGKQTGFLFYVPAWNTSKIDPETGFVDLLKPRYENIAQSQAFFGKFDKICYNTDKGYFEFHIDYAKFTDKAKNSRQKWAICSHGDKRYVYDKTANQNKGAAKGINVNDELKSLFARYHINDKQPNLVMDICQNNDKEFHKSLMCLLKTLLALRYSNASSDEDFILSPVANDEGVFFNSALADDTQPQNADANGAYHIALKGLWLLNELKNSDDLNKVKLAIDNQTWLNFAQNR Prevotella bryantii SEQMKFTDFTGLYSLSKTLRFELKPIGKTLENIKKAGLLEQDQHRADSY (Pb2Cas12a) IDKKVKKIIDEYHKAFIEKSLSNFELKYQSEDKLDSLEEYLMYYSMKR NCBI Gene ID: NO:IEKTEKDKFAKIQDNLRKQIADHLKGDESYKTIFSKDLIRKNLPDFV WP_039871282.1 11KSDEERTLIKEFKDFTTYFKGFYENRENMYSAEDKSTAISHRIIHENLPKFVDNINAFSKIILIPELREKLNQIYQDFEEYLNVESIDEIFHLDYFSMVMTQKQIEVYNAIIGGKSTNDKKIQGLNEYINLYNQKHKDCKLPKLKLLFKQILSDRIAISWLPDNFKDDQEALDSIDTCYKNLLNDGNVLGEGNLKLLLENIDTYNLKGIFIRNDLQLTDISQKMYASWNVIQDAVILDLKKQVSRKKKESAEDYNDRLKKLYTSQESFSIQYLNDCLRAYGKTENIQDYFAKLGAVNNEHEQTINLFAQVRNAYTSVQAILTTPYPENANLAQDKETVALIKNLLDSLKRLQRFIKPLLGKGDESDKDERFYGDFTPLWETLNQITPLYNMVRNYMTRKPYSQEKIKLNFENSTLLGGWDLNKEHDNTAIILRKNGLYYLAIMKKSANKIFDKDKLDNSGDCYEKMVYKLLPGANKMLPKVFFSKSRIDEFKPSENIIENYKKGTHKKGANFNLADCHNLIDFFKSSISKHEDWSKFNFHFSDTSSYEDLSDFYREVEQQGYSISFCDVSVEYINKMVEKGDLYLFQIYNKDFSEFSKGTPNMHTLYWNSLFSKENLNNIIYKLNGQAEIFFRKKSLNYKRPTHPAHQAIKNKNKCNEKKESIFDYDLVKDKRYTVDKFQFHVPITMNFKSTGNTNINQQVIDYLRTEDDTHIIGIDRGERHLLYLVVIDSHGKIVEQFTLNEIVNEYGGNIYRTNYHDLLDTREQNREKARESWQTIENIKELKEGYISQVIHKITDLMQKYHAVVVLEDLNMGFMRGRQKVEKQVYQKFEEMLINKLNYLVNKKADQNSAGGLLHAYQLTSKFESFQKLGKQSGFLFYIPAWNTSKIDPVTGFVNLFDTRYESIDKAKAFFGKFDSIRYNADKDWFEFAFDYNNFTTKAEGTRTNWTICTYGSRIRTFRNQAKNSQWDNEEIDLTKAYKAFFAKHGINIYDNIKEAIAMETEKSFFEDLLHLLKLTLQMRNSITGTTTDYLISPVHDSKGNFYDSRICDNSLPANADANGAYNIARKGLMLIQQIKDSTSSNRFKFSPITNKDWLIFAQEK PYLND Candidatus SEQMENKNNQTQSIWSVFTKKYSLQKTLRFELKPVGETKKWLEENDIF Parcubacteria IDKKDLNIDKSYNQAKFYFDKLHQDFIKESLSVENGIRNIDFEKFAKIF bacterium NO:ESNKEKIVSLKKKNKEVKDKNKKNWDEISKLEKEIEGQRENLYKEI (PgCas12a) 12RELFDKRAEKWKKEYQDKEIERGGKKEKIKFSSADLKQKGVNFLT NCBI Gene ID:AAGIINILKYKFPAEKDEEFRKEGYPSLFINDELNPGKKIYIFESFDK BCX15829.1FTTYLSKFQQTRENLYKDDGTSTAVATRIVSNFERFLENKSLFEEKYKNKAKDVGLTKEEEKVFEINYYYDCLIQEGIDKYNKIIGEINRKTKEYRDKNKIDKKDLPLFLNLEKQILGEVKKERVFIEAKDEKTEEEVFIDRFQEFIKRNKIKIYGDEKEEIEGAKKFIEDFTSGIFENDYQSIYLKKNVINEIVNKWFSNPEEFLMKLTGVKSEEKIKLKKFTSLDEFKNAILSLEGDIFKSRFYKNEVNPEAPLEKEEKSNNWENFLKIWRFEFESLFKDKVEKGEIKKDKNGEPIQIFWGYTDKLEKEAEKIKFYSAEKEQIKTIKNYCDAALRINRMMRYFNLSDKDRKDVPSGLSTEFYRLVDEYFNNFEFNKYYNGIRNFITKKPSDENKIKLNFESRSLLDGWDVSKEKDNLGLIFIKNNKYYLGVLRKENSKLFDYQITEKDNQKEKERKNNLKNEILANDNEDFYLKMNYWQIADPAKDIFNLVLMPDNTVKRFTKLEEKNKHWPDEIKRIKEKGTYKREKVNREDLVKIINYFRKCALIYWKKFDLKLLPSEEYQTFKDFTDHIALQGYKINFDKIKASYIEKQLNDGNLYLFEVSNKDFYKYKKPDSRKNIHTLYWEHIFSKENLEEIKYPLIRLNGKAEIFYRDVLEMNEEMRKPVILERLNGAKQAKREDKPVYHYQRYLKPTYLFHCPITLNADKPSSSFKNFSSKLNHFIKDNLGKINIIGIDRGEKNLLYYCVINQNQEILDYGSLNKINLNKVNNVNYFDKLVEREKQRQLERQSWEPVAKIKDLKQGYISYVVRKICDLIINHNAIVVLEDLSRRFKQIRNGISERTVYQQFEKALIDKLNYLIFKDNRDVFSPGGVLNGYQLAAPFTSFKDIEKAKQTGVLFYTSAEYTSQTDPLTGFRKNIYISNSASQEKIKELINKLKKFGWDDTEESYFIEYNQVDFAEKKKKPLSKDWTIWTKVPRVIRWKESKSSYWSYKKINLNEEFRDLLEKYGFEAQSNDILSNLKKRIAENDKLLVEKKEFDGRLKNFYERFIFLFNIVLQVRNTYSLSVEIDKTEKKLKKIDYGIDFFASPVKPFFTTFGLREIGIEKDGKVVKDNAREEIASENLAEFKDRLKEYKPEEKFDADGVGAYNIARKGLIILEKIKNNPNKPDLSISKEEWDKFVQR Acidaminococcus SEQMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDH sp. IDYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETR (AaCas12a) NO:NALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFN NCBI Gene ID: 13GKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDI WP_021736722.1STAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISN QDWLAYIQELRN BacteroidetesSEQ MESPTTQLKKFTNLYQLSKTLRFELKPVGKTKEHIETKGILKKDEE bacterium IDRAVNYKLIKKIIDGFHKHFIELAMQQVKLSKLDELAELYNASAERK (BoCas12a) NO:KEESYKKELEQVQAALRKEIVKGFNIGEAKEIFSKIDKKELFTELLD NCBI Gene ID: 14EWVKNLEEKKLVDDFKTFTTYFTGFHENRKNMYTDKAQSTAIAY PKP47250.1RLVHENLPKFLDNTKIFKQIETKFEASKIEEIETKLEPIIQGTSLSEIFTLDYYNHALTQAGIDFINNIIGGYTEDEGKKKIQGLNEYINLYNQKQEKKNRIPKLKILYKQILSDRDSISFLPDAFEDSQEVLNAIQNYYQTNLIDFKPKDKEETENVLEETKKLLTELFSNELSKIYIRNDKAITDISQALFNDWGVFKSALEYKFIQDLELGTKELSKKQENEKEKYLKQAYFSIAEIENALFAYQNETDVLNEIKENSHPIADYFTKHFKAKKKVDTSTSSVEKDFDLIANIDAKYSCIKGILNTDYPKDKKLNQEKKTIDDLKVFLDSLMELLHFVKPLALPNDSILEKDENFYSHFESYYEQLELLIPLYNKVRNYAAKKPYSTEKFKLNFENATLLKGWDKNKEIDNTSVILRKRGLYYLAIMPQDNKNVFKKSPNLKNNESCFEKMDYKQMALPMGFGAFVRKCFGTAFQLGWNCPKSCINEEDKIIIKEDEVKNNRAEIIDCYKDFLNIYEKDGFQYKEYGFNFKESKEYESLREFFIDVEQKGYKIEFQNISENYIHQLVNEGKLYLFQIYNKDFSSYSKGKPNMHTMYWKALFDPENLKDVVYKLNGQAEVFYRKKSIEDKNIITHKANEPIENKNPKAKKTQSTFEYDLIKDKRYTVDKFHFHVPITINFKATGNNYINQQVLDHLKNNTDVNIIGLDRGERHLIYLTLINQKGEILLQESLNTIVNKKFDIETPYHTLLQNKEDERAKARENWGVIENIKELKEGYLSQVVHKIAKLMVDYNAIVVMEDLNTGFKRGRFKVEKQVYQKLEKMLIDKLNYLVFKDKDPNEVGGLYNALQLTNKFESFSKMGKQSGFLFYVPAWNTSKIDPTTGFVNLFYAKYESIPKAQDFFTKFKSIRYNSDENYFEFAFDYNDFTTRAEGTKSDWTVCTYGDRIKTFRNPEKNNQWDNQEVNLIEQFEAFFGKHNITYGDGNCIKKQLIEQDKKEFFEELFHLFKLTLQMRNSITNSEIDYLISPVKNSKKEFYDSRKADSTLPKDADANGAYHIAKKGLMWLEKINSFKGSDWKKLDLDKTNKTWLNFVQETASEKHKKLQTV Candidatus SEQMDAKEFTGQYPLSKTLRFELRPIGRTWDNLEASGYLAEDRHRAEC Methanomethyl- IDYPRAKELLDDNHRAFLNRVLPQIDMDWHPIAEAFCKVHKNPGNK ophilus alvus NO:ELAQDYNLQLSKRRKEISAYLQDADGYKGLFAKPALDEAMKIAKE Mx1201 15NGNESDIEVLEAFNGFSVYFTGYHESRENIYSDEDMVSVAYRITED (CMaCas12a)NFPRFVSNALIFDKLNESHPDIISEVSGNLGVDDIGKYFDVSNYNNF NCBI Gene ID:LSQAGIDDYNHIIGGHTTEDGLIQAFNVVLNLRHQKDPGFEKIQFK 15139718QLYKQILSVRTSKSYIPKQFDNSKEMVDCICDYVSKIEKSETVERALKLVRNISSFDLRGIFVNKKNLRILSNKLIGDWDAIETALMHSSSSENDKKSVYDSAEAFTLDDIFSSVKKFSDASAEDIGNRAEDICRVISETAPFINDLRAVDLDSLNDDGYEAAVSKIRESLEPYMDLFHELEIFSVGDEFPKCAAFYSELEEVSEQLIEIIPLFNKARSFCTRKRYSTDKIKVNLKFPTLADGWDLNKERDNKAAILRKDGKYYLAILDMKKDLSSIRTSDEDESSFEKMEYKLLPSPVKMLPKIFVKSKAAKEKYGLTDRMLECYDKGMHKSGSAFDLGFCHELIDYYKRCIAEYPGWDVFDFKFRETSDYGSMKEFNEDVAGAGYYMSLRKIPCSEVYRLLDEKSIYLFQIYNKDYSENAHGNKNMHTMYWEGLFSPQNLESPVFKLSGGAELFFRKSSIPNDAKTVHPKGSVLVPRNDVNGRRIPDSIYRELTRYFNRGDCRISDEAKSYLDKVKTKKADHDIVKDRRFTVDKMMFHVPIAMNFKAISKPNLNKKVIDGIIDDQDLKIIGIDRGERNLIYVTMVDRKGNILYQDSLNILNGYDYRKALDVREYDNKEARRNWTKVEGIRKMKEGYLSLAVSKLADMIIENNAIIVMEDLNHGFKAGRSKIEKQVYQKFESMLINKLGYMVLKDKSIDQSGGALHGYQLANHVTTLASVGKQCGVIFYIPAAFTSKIDPTTGFADLFALSNVKNVASMREFFSKMKSVIYDKAEGKFAFTFDYLDYNVKSECGRTLWTVYTVGERFTYSRVNREYVRKVPTDIIYDALQKAGISVEGDLRDRIAESDGDTLKSIFYAFKYALDMRVENREED YIQSPVKNASGEFFCSKNAGKSLPQDSDANGAYNIALKGILQLRMLSEQYDPNAESIRLPLITNKAWLTFMQSGMKTWKN

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with LbCas12a): K538A, K538D, K538E, Y542A, Y542D, Y542E, orK595A, K595D, K595E relative to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with AsCas12a): K548A, K548D, K548E, N552A, N552D, N552E, orK607A, K607D, K607 relative to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with CtCas12a): K534A, K534D, K534E, Y538A, Y538D, Y538E, orR591A, R591D, R591E relative to the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with EeCas12a): K542A, K541D, K541E, N545A, N545D, N545E orK601A, K601D, K601E relative to the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with Mb3Cas12a): K579A, K579D, K579E, N583A, N583D, N583E orK635A, K635D, K635E relative to the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with FnCas12a): K613A, K613D, K613E, N617A, N617D, N617E orK671A, K671D, K671E relative to the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with FnoCas12a): K613A, K613D, K613E, N617A, N617D, N617E orN671A, N671D, N671E relative to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with FbCas12a): K617A, K617D, K617E, N621A, N621D, N621E orK678A, K678D, K678E relative to the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with Lb4Cas12a): K541A, K541D, K541E, N545A, N545D, N545E orK601A, K601D, K601E relative to the amino acid sequence of SEQ ID NO: 9.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with MbCas12a): K569A, K569D, K569E, N573A, N573D, N573E orK625A, K625D, K625E relative to the amino acid sequence of SEQ ID NO:10.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with Pb2Cas12a): K562A, K562D, K562E, N566A, N566D, N566E orK619A, K619D, K619E relative to the amino acid sequence of SEQ ID NO:11.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with PgCas12a): K645A, K645D, K645E, N649A, N649D, N649E orK732A, K732D, K732E relative to the amino acid sequence of SEQ ID NO:12.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with AaCas12a): K548A, K548D, K548E, N552A, N552D, N552E orK607A, K607D, K607E relative to the amino acid sequence of SEQ ID NO:13.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with BoCas12a): K592A, K592D, K592E, N596A, N596D, N596E orK653A, K653D, K653E relative to the amino acid sequence of SEQ ID NO:14.

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease contains one or more of the following substitutions(aligned with CMaCas12a): K521A, K521D, K521E, K525A, K525D, K525E orK577A, K577D, K577E relative to the amino acid sequence of SEQ ID NO:15.

The mutations described herein may be described in the context of anatural Cas12a (any one of SEQ ID NOs: 15) sequence and mutationalpositions can be carried out by aligning the amino acid sequence of aCas12a nucleic acid-guided nuclease with, for example, SEQ ID NO: 1 andmaking the equivalent modification (e.g., substitution) at theequivalent position. By way of example, Table 8 illustrates theequivalent amino acid positions of fifteen orthologous Cas12a nucleicacid-guided nucleases (SEQ ID NOs: 1-15). Any one of the amino acidsindicated in Table 8 may be mutated (i.e., via a comparable amino acidsubstitution).

TABLE 8 Equivalent amino acid positions in homologous Cas12a nucleicacid-guided nuclease Cas 12a AA AA AA AA WT SEQ ID NO Ortholog positionposition position position SEQ ID NO: 1  LbCas12a G532 K538 Y542 K595SEQ ID NO: 2  AsCas12a S542 K548 N552 K607 SEQ ID NO: 3  CtCas12a N528K534 Y538 R591 SEQ ID NO: 4  EeCas12a N535 K541 N545 K601 SEQ ID NO: 5 Mb3Cas12a N573 K579 N583 K635 SEQ ID NO: 6  FnCas12a N607 K613 N617 K671SEQ ID NO: 7  FnoCas12a N607 K613 N617 N671 SEQ ID NO: 8  FbCas12a N611K617 N621 K678 SEQ ID NO: 9  Lb4Cas12a N535 K541 N545 K601 SEQ ID NO: 10MbCas12a N563 K569 N573 K625 SEQ ID NO: 11 Pb2Cas12a G556 K562 N566 K619SEQ ID NO: 12 PgCas12a D639 K645 N649 K732 SEQ ID NO: 13 AaCas12a S542K548 N552 K607 SEQ ID NO: 14 BoCas12a K586 K592 N596 K653 SEQ ID NO: 15CMaCas12a D515 K521 N525 K577

The variant single-strand-specific Cas12a nucleic acid-guided nucleasesof the disclosure may have at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to any one of SEQ ID NOs:1-15 (excluding the residues listed in Table 8) and contain anyconservative mutation one or more residues indicated in Tables 9-13.

It should be appreciated that any of the amino acid mutations describedherein, (e.g., K595A) from a first amino acid residue (e.g., K, an aminoacid with a basic side chain) to a second amino acid residue (e.g., A,an amino acid with an aliphatic side chain) may also include mutationsfrom the first amino acid residue, lysine, to an amino acid residue thatis similar to (e.g., conserved) the second amino acid residue, alanine,such as valine or glycine. As another example, mutation of an amino acidwith a positively charged side chain (e.g., arginine, histidine, orlysine) may be a mutation to a second amino acid with an acidic sidechain (e.g., glutamic acid or aspartic acid). As another example,mutation of an amino acid with a polar side chain (e.g., serine,threonine, asparagine, or glutamine) may be a mutation to a second aminoacid with a positively charged side chain (e.g., arginine, histidine, orlysine). The skilled artisan would recognize that such conservativeamino acid substitutions will likely have minor effects on proteinstructure and are likely to be well tolerated without compromisingfunction. That is, a mutation from one amino acid to a threonine may bean amino acid mutation to a serine; a mutation from one amino acid to anarginine may be an amino acid mutation to a lysine; a mutation from oneamino acid to an isoleucine, may be an amino acid mutation to analanine, valine, methionine, or leucine; a mutation from one amino acidto a lysine may be an amino acid mutation to an arginine; a mutationfrom one amino acid to an aspartic acid may be an amino acid mutation toa glutamic acid or asparagine; a mutation from one amino acid to avaline may be an amino acid mutation to an alanine, isoleucine,methionine, or leucine; a mutation from one amino acid to a glycine maybe an amino acid mutation to an alanine. It should be appreciated,however, that additional conserved amino acid residues would berecognized by the skilled artisan and any of the amino acid mutations toother conserved amino acid residues are also within the scope of thisdisclosure.

Exemplary variant Cas12a orthologs are shown in tables 9-13.

TABLE 9 Exemplary Variant Ortholog Cas12a’s Variant LbCas12a VariantAsCas12a Variant CtCas12a SEQ (in relation to wt SEQ (in relation to wtSEQ (in relation to wt ID LbCas12a SEQ ID ID AsCas12a SEQ ID ID CtCas12aSEQ ID NO: NO: 1) NO: NO: 2) NO: NO: 3) 16 K595A 55 K607A  94 R591A 17K595D 56 K607D  95 R591D 18 K595E 57 K607E  96 R591E 19 K538A/K595A 58K548A/K607A  97 K534A/R591A 20 K538A/K595D 59 K548A/K607D  98K534A/R591D 21 K538A/K595E 60 K548A/K607E  99 K534A/R591E 22 K538D/K595A61 K548D/K607A 100 K534D/R591A 23 K538D/K595D 62 K548D/K607D 101K534D/R591D 24 K538D/K595E 63 K548D/K607E 102 K534D/R591E 25 K538E/K595A64 K548E/K607A 103 K534E/R591A 26 K538E/K595D 65 K548E/K607D 104K534E/R591D 27 K538E/K595E 66 K548E/K607E 105 K534E/R591E 28K538A/Y542A/K595A 67 K548A/N552A/K607A 106 K534A/Y538A/R591A 29K538A/Y542D/K595A 68 K548A/N552D/K607A 107 K534A/Y538D/R591A 30K538A/Y542E/K595A 69 K548A/N552E/K607A 108 K534A/Y538E/R591A 31K538A/Y542A/K595D 70 K548A/N552A/K607D 109 K534A/Y538A/R591D 32K538A/Y542D/K595D 71 K548A/N552D/K607D 110 K534A/Y538D/R591D 33K538A/Y542E/K595D 72 K548A/N552E/K607D 111 K534A/Y538E/R591D 34K538A/Y542A/K595E 73 K548A/N552A/K607E 112 K534A/Y538A/R591E 35K538A/Y542D/K595E 74 K548A/N552D/K607E 113 K534A/Y538D/R591E 36K538A/Y542E/K595E 75 K548A/N552E/K607E 114 K534A/Y538E/R591E 37K538D/Y542A/K595A 76 K548D/N552A/K607A 115 K534D/Y538A/R591A 38K538D/Y542D/K595A 77 K548D/N552D/K607A 116 K534D/Y538D/R591A 39K538D/Y542E/K595A 78 K548D/N552E/K607A 117 K534D/Y538E/R591A 40K538D/Y542A/K595D 79 K548D/N552A/K607D 118 K534D/Y538A/R591D 41K538D/Y542D/K595D 80 K548D/N552D/K607D 119 K534D/Y538D/R591D 42K538D/Y542E/K595D 81 K548D/N552E/K607D 120 K534D/Y538E/R591D 43K538D/Y542A/K595E 82 K548D/N552A/K607E 121 K534D/Y538A/R591E 44K538D/Y542D/K595E 83 K548D/N552D/K607E 122 K534D/Y538D/R591E 45K538D/Y542E/K595E 84 K548D/N552E/K607E 123 K534D/Y538E/R591E 46K538E/Y542A/K595A 85 K548E/N552A/K607A 124 K534E/Y538A/R591A 47K538E/Y542D/K595A 86 K548E/N552D/K607A 125 K534E/Y538D/R591A 48K538E/Y542E/K595A 87 K548E/N552E/K607A 126 K534E/Y538E/R591A 49K538E/Y542A/K595E 88 K548E/N552A/K607D 127 K534E/Y538A/R591D 50K538E/Y542D/K595E 89 K548E/N552D/K607D 128 K534E/Y538D/R591D 51K538E/Y542E/K595E 90 K548E/N552E/K607D 129 K534E/Y538E/R591D 52K538E/Y542A/K595E 91 K548E/N552A/K607E 130 K534E/Y538A/R591E 53K538E/Y542D/K595E 92 K548E/N552D/K607E 131 K534E/Y538D/R591E 54K538E/Y542E/K595E 93 K548E/N552E/K607E 132 K534E/Y538E/R591E

TABLE 10 Exemplary Variant Ortholog Cas12a’s Variant EeCas12a SEQ (inrelation to wt ID EeCas12a SEQ ID NO: NO: 4) 133 K601A 134 K601D 135K601E 136 K541A/K601A 137 K541A/K601D 138 K541A/K601E 139 K541D/K601A140 K541D/K601D 141 K541D/K601E 142 K541E/K601A 143 K541E/K601D 144K541E/K601E 145 K541A/N545A/K601A 146 K541A/N545D/K601A 147K541A/N545E/K601A 148 K541A/N545A/K601D 149 K541A/N545D/K601D 150K541A/N545E/K601D 151 K541A/N545A/K601E 152 K541A/N545D/K601E 153K541A/N545E/K601E 154 K541D/N545A/K601A 155 K541D/N545D/K601A 156K541D/N545E/K601A 157 K541D/N545A/K601D 158 K541D/N545D/K601D 159K541D/N545E/K601D 160 K541D/N545A/K601E 161 K541D/N545D/K601E 162K541D/N545E/K601E 163 K541E/N545A/K601A 164 K541E/N545D/K601A 165K541E/N545E/K601A 166 K541E/N545A/K601D 167 K541E/N545D/K601D 168K541E/N545E/K601D 169 K541E/N545A/K601E 170 K541E/N545D/K601E 171K541E/N545E/K601E 172 K635A 173 K635D 174 K635E 175 K579A/K635A 176K579A/K635D 177 K579A/K635E 178 K579D/K635A 179 K579D/K635D 180K579D/K635E 181 K579E/K635A 182 K579E/K635D 183 K579E/K635E 184K579A/N583A/K635A 185 K579A/N583D/K635A 186 K579A/N583E/K635A 187K579A/N583A/K635D 188 K579A/N583D/K635D 189 K579A/N583E/K635D 190K579A/N583A/K635E 191 K579A/N583D/K635E 192 K579A/N583E/K635E 193K579D/N583A/K635A 194 K579D/N583D/K635A 195 K579D/N583E/K635A 196K579D/N583A/K635D 197 K579D/N583D/K635D 198 K579D/N583E/K635D 199K579D/N583A/K635E 200 K579D/N583D/K635E 201 K579D/N583E/K635E 202K579E/N583A/K635A 203 K579E/N583D/K635A 204 K579E/N583E/K635A 205K579E/N583A/K635D 206 K579E/N583D/K635D 207 K579E/N583E/K635D 208K579E/N583A/K635E 209 K579E/N583D/K635E 210 K579E/N583E/K635E 211 K671A212 K671D 213 K671E 214 K613A/K671A 215 K613A/K671D 216 K613A/K671E 217K613D/K671A 218 K613D/K671D 219 K613D/K671E 220 K613E/K671A 221K613E/K671D 222 K613E/K671E 223 K613A/N617A/K671A 224 K613A/N617D/K671A225 K613A/N617E/K671A 226 K613A/N617A/K671D 227 K613A/N617D/K671D 228K613A/N617E/K671D 229 K613A/N617A/K671E 230 K613A/N617D/K671E 231K613A/N617E/K671E 232 K613D/N617A/K671A 233 K613D/N617D/K671A 234K613D/N617E/K671A 235 K613D/N617A/K671D 236 K613D/N617D/K671D 237K613D/N617E/K671D 238 K613D/N617A/K671E 239 K613D/N617D/K671E 240K613D/N617E/K671E 241 K613E/N617A/K671A 242 K613E/N617D/K671A 243K613E/N617E/K671A 244 K613E/N617A/K671D 245 K613E/N617D/K671D 246K613E/N617E/K671D 247 K613E/N617A/K671E 248 K613E/N617D/K671E 249K613E/N617E/K671E

TABLE 11 Exemplary Variant Ortholog Cas12a’s SEQ Variant FnoCas12a ID(in relation to wt NO: FnoCas12a SEQ ID NO: 7) 250 N671A 251 N671D 252N671E 253 K613A/N671A 254 K613A/N671D 255 K613A/N671E 256 K613D/N671A257 K613D/N671D 258 K613D/N671E 259 K613E/N671A 260 K613E/N671D 261K613E/N671E 262 K613A/N617A/N671A 263 K613A/N617D/N671A 264K613A/N617E/N671A 265 K613A/N617A/N671D 266 K613A/N617D/N671D 267K613A/N617E/N671D 268 K613A/N617A/N671E 269 K613A/N617D/N671E 270K613A/N617E/N671E 271 K613D/N617A/N671A 272 K613D/N617D/N671A 273K613D/N617E/N671A 274 K613D/N617A/N671D 275 K613D/N617D/N671D 276K613D/N617E/N671D 277 K613D/N617A/N671E 278 K613D/N617D/N671E 279K613D/N617E/N671E 280 K613E/N617A/N671A 281 K613E/N617D/N671A 282K613E/N617E/N671A 283 K613E/N617A/N671D 284 K613E/N617D/N671D 285K613E/N617E/N671D 286 K613E/N617A/N671E 287 K613E/N617D/N671E 288K613E/N617E/N671E 289 K678A 290 K678D 291 K678E 292 K617A/K678A 293K617A/K678D 294 K617A/K678E 295 K617D/K678A 296 K617D/K678D 297K617D/K678E 298 K617E/K678A 299 K617E/K678D 300 K617E/K678E 301K617A/N621A/K678A 302 K617A/N621D/K678A 303 K617A/N621E/K678A 304K617A/N621A/K678D 305 K617A/N621D/K678D 306 K617A/N621E/K678D 307K617A/N621A/K678E 308 K617A/N621D/K678E 309 K617A/N621E/K678E 310K617D/N621A/K678A 311 K617D/N621D/K678A 312 K617D/N621E/K678A 313K617D/N621A/K678D 314 K617D/N621D/K678D 315 K617D/N621E/K678D 316K617D/N621A/K678E 317 K617D/N621D/K678E 318 K617D/N621E/K678E 319K617E/N621A/K678A 320 K617E/N621D/K678A 321 K617E/N621E/K678A 322K617E/N621A/K678D 323 K617E/N621D/K678D 324 K617E/N621E/K678D 325K617E/N621A/K678E 326 K617E/N621D/K678E 327 K617E/N621E/K678E 328 K601A329 K601D 330 K601E 331 K541A/K601A 332 K541A/K601D 333 K541A/K601E 334K541D/K601A 335 K541D/K601D 336 K541D/K601E 337 K541E/K601A 338K541E/K601D 339 K541E/K601E 340 K541A/N545A/K601A 341 K541A/N545D/K601A342 K541A/N545E/K601A 343 K541A/N545A/K601D 344 K541A/N545D/K601D 345K541A/N545E/K601D 346 K541A/N545A/K601E 347 K541A/N545D/K601E 348K541A/N545E/K601E 349 K541D/N545A/K601A 350 K541D/N545D/K601A 351K541D/N545E/K601A 352 K541D/N545A/K601D 353 K541D/N545D/K601D 354K541D/N545E/K601D 355 K541D/N545A/K601E 356 K541D/N545D/K601E 357K541D/N545E/K601E 358 K541E/N545A/K601A 359 K541E/N545D/K601A 360K541E/N545E/K601A 361 K541E/N545A/K601D 362 K541E/N545D/K601D 363K541E/N545E/K601D 364 K541E/N545A/K601E 365 K541E/N545D/K601E 366K541E/N545E/K601E

TABLE 12 Exemplary Variant Ortholog Cas12a’s SEQ Variant MbCas12a ID (inrelation to wt NO: MbCas12a SEQ ID NO: 10) 367 K625A 368 K625D 369 K625E370 K569A/K625A 371 K569A/K625D 372 K569A/K625E 373 K569D/K625A 374K569D/K625D 375 K569D/K625E 376 K569E/K625A 377 K569E/K625D 378K569E/K625E 379 K569A/N573A/K625A 380 K569A/N573D/K625A 381K569A/N573E/K625A 382 K569A/N573A/K625D 383 K569A/N573D/K625D 384K569A/N573E/K625D 385 K569A/N573A/K625E 386 K569A/N573D/K625E 387K569A/N573E/K625E 388 K569D/N573A/K625A 389 K569D/N573D/K625A 390K569D/N573E/K625A 391 K569D/N573A/K625D 392 K569D/N573D/K625D 393K569D/N573E/K625D 394 K569D/N573A/K625E 395 K569D/N573D/K625E 396K569D/N573E/K625E 397 K569E/N573A/K625A 398 K569E/N573D/K625A 399K569E/N573E/K625A 400 K569E/N573A/K625D 401 K569E/N573D/K625D 402K569E/N573E/K625D 403 K569E/N573A/K625E 404 K569E/N573D/K625E 405K569E/N573E/K625E 406 K619A 407 K619D 408 K619E 409 K562A/K619A 410K562A/K619D 411 K562A/K619E 412 K562D/K619A 413 K562D/K619D 414K562D/K619E 415 K562E/K619A 416 K562E/K619D 417 K562E/K619E 418K562A/N566A/K619A 419 K562A/N566D/K619A 420 K562A/N566E/K619A 421K562A/N566A/K619D 422 K562A/N566D/K619D 423 K562A/N566E/K619D 424K562A/N566A/K619E 425 K562A/N566D/K619E 426 K562A/N566E/K619E 427K562D/N566A/K619A 428 K562D/N566D/K619A 429 K562D/N566E/K619A 430K562D/N566A/K619D 431 K562D/N566D/K619D 432 K562D/N566E/K619D 433K562D/N566A/K619E 434 K562D/N566D/K619E 435 K562D/N566E/K619E 436K562E/N566A/K619A 437 K562E/N566D/K619A 438 K562E/N566E/K619A 439K562E/N566A/K619D 440 K562E/N566D/K619D 441 K562E/N566E/K619D 442K562E/N566A/K619E 443 K562E/N566D/K619E 444 K562E/N566E/K619E 445 K732A446 K732D 447 K732E 448 K645A/K732A 449 K645A/K732D 450 K645A/K732E 451K645D/K732A 452 K645D/K732D 453 K645D/K732E 454 K645E/K732A 455K645E/K732D 456 K645E/K732E 457 K645A/N649A/K732A 458 K645A/N649D/K732A459 K645A/N649E/K732A 460 K645A/N649A/K732D 461 K645A/N649D/K732D 462K645A/N649E/K732D 463 K645A/N649A/K732E 464 K645A/N649D/K732E 465K645A/N649E/K732E 466 K645D/N649A/K732A 467 K645D/N649D/K732A 468K645D/N649E/K732A 469 K645D/N649A/K732D 470 K645D/N649D/K732D 471K645D/N649E/K732D 472 K645D/N649A/K732E 473 K645D/N649D/K732E 474K645D/N649E/K732E 475 K645E/N649A/K732A 476 K645E/N649D/K732A 477K645E/N649E/K732A 478 K645E/N649A/K732D 479 K645E/N649D/K732D 480K645E/N649E/K732D 481 K645E/N649A/K732E 482 K645E/N649D/K732E 483K645E/N649E/K732E

TABLE 13 Exemplary Variant Ortholog Cas12a’s SEQ Variant AaCas12a ID (inrelation to wt NO: AaCas12a SEQ ID NO: 13) 484 K607A 485 K607D 486 K607E487 K548A/K607A 488 K548A/K607D 489 K548A/K607E 490 K548D/K607A 491K548D/K607D 492 K548D/K607E 493 K548E/K607A 494 K548E/K607D 495K548E/K607E 496 K548A/N552A/K607A 497 K548A/N552D/K607A 498K548A/N552E/K607A 499 K548A/N552A/K607D 500 K548A/N552D/K607D 501K548A/N552E/K607D 502 K548A/N552A/K607E 503 K548A/N552D/K607E 504K548A/N552E/K607E 505 K548D/N552A/K607A 506 K548D/N552D/K607A 507K548D/N552E/K607A 508 K548D/N552A/K607D 509 K548D/N552D/K607D 510K548D/N552E/K607D 511 K548D/N552A/K607E 512 K548D/N552D/K607E 513K548D/N552E/K607E 514 K548E/N552A/K607A 515 K548E/N552D/K607A 516K548E/N552E/K607A 517 K548E/N552A/K607D 518 K548E/N552D/K607D 519K548E/N552E/K607D 520 K548E/N552A/K607E 521 K548E/N552D/K607E 522K548E/N552E/K607E 523 K653A 524 K653D 525 K653E 526 K592A/K653A 527K592A/K653D 528 K592A/K653E 529 K592D/K653A 530 K592D/K653D 531K592D/K653E 532 K592E/K653A 533 K592E/K653D 534 K592E/K653E 535K592A/N596A/K653A 536 K592A/N596D/K653A 537 K592A/N596E/K653A 538K592A/N596A/K653D 539 K592A/N596D/K653D 540 K592A/N596E/K653D 541K592A/N596A/K653E 542 K592A/N596D/K653E 543 K592A/N596E/K653E 544K592D/N596A/K653A 545 K592D/N596D/K653A 546 K592D/N596E/K653A 547K592D/N596A/K653D 548 K592D/N596D/K653D 549 K592D/N596E/K653D 550K592D/N596A/K653E 551 K592D/N596D/K653E 552 K592D/N596E/K653E 553K592E/N596A/K653A 554 K592E/N596D/K653A 555 K592E/N596E/K653A 556K592E/N596A/K653D 557 K592E/N596D/K653D 558 K592E/N596E/K653D 559K592E/N596A/K653E 560 K592E/N596D/K653E 561 K592E/N596E/K653E 562 K577A563 K577D 564 K577E 565 K521A/K577A 566 K521A/K577D 567 K521A/K577E 568K521D/K577A 569 K521D/K577D 570 K521D/K577E 571 K521E/K577A 572K521E/K577D 573 K521E/K577E 574 K521A/N525A/K577A 575 K521A/N525D/K577A576 K521A/N525E/K577A 577 K521A/N525A/K577D 578 K521A/N525D/K577D 579K521A/N525E/K577D 580 K521A/N525A/K577E 581 K521A/N525D/K577E 582K521A/N525E/K577E 583 K521D/N525A/K577A 584 K521D/N525D/K577A 585K521D/N525E/K577A 586 K521D/N525A/K577D 587 K521D/N525D/K577D 588K521D/N525E/K577D 589 K521D/N525A/K577E 590 K521D/N525D/K577E 591K521D/N525E/K577E 592 K521E/N525A/K577A 593 K521E/N525D/K577A 594K521E/N525E/K577A 595 K521E/N525A/K577D 596 K521E/N525D/K577D 597K521E/N525E/K577D 598 K521E/N525A/K577E 599 K521E/N525D/K577E 600K521E/N525E/K577E

In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease is at least 70% identical to any one of SEQ ID NOs:16-600. In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease is at least 75% identical to any one of SEQ ID NOs:16-600 16-600. In some embodiments, the single-strand-specific Cas12anucleic acid-guided nuclease is at least 80% identical to any one of SEQID NOs: 16-600. In some embodiments, the single-strand-specific Cas12anucleic acid-guided nuclease is at least 85% identical to any one of SEQID NOs: 16-600. In some embodiments, the single-strand-specific Cas12anucleic acid-guided nuclease is at least 90% identical to any one of SEQID NOs: 16-600. In some embodiments, the single-strand-specific Cas12anucleic acid-guided nuclease is at least 95% identical to any one of SEQID NOs: 16-600. In some embodiments, the single-strand-specific Cas12anucleic acid-guided nuclease is at least 96%, 97%, 98% or 99% identicalto any one of SEQ ID NOs: 16-600. In some embodiments, thesingle-strand-specific Cas12a nucleic acid-guided nuclease is any one ofSEQ ID NOs: 16-600.

The mutations described herein are described in the context of the WTLbCas12a (e.g., SEQ ID NO: 1) sequence and mutational positions can becarried out by aligning the amino acid sequence of a Cas12a nucleicacid-guided nuclease with SEQ ID NO: 1 and making the equivalentmodification (e.g., substitution) at the equivalent position. By way ofexample, the mutations described herein may be applied to a Cas12aenzyme shown in Table 7, or any other homolog Cas12a thereof by aligningthe amino acid sequence of the Cas12a to SEQ ID NO: 1 and making themodifications described in Tables 9-13 (changes to the wildtype residueto alanine, aspartic acid or glutamic acid or conservative equivalentsat the Cas12a ortholog's equivalent position (e.g., see Table 8 for anexample of equivalent residue positions).

For example, in addition to the variant LbCas12a sequences in Table 9(variant sequences SEQ ID Nos: 16-54), like variants are envisioned forAsCas12a (variant sequences SEQ ID Nos: 55-93), CtCas12a (variantsequences SEQ ID Nos: 94-132), EeCas12a (variant sequences SEQ ID Nos:133-171), Mb3Cas12a (variant sequences SEQ ID Nos: 172-210), FnCas12a(variant sequences SEQ ID Nos: 211-249), FnoCas12a (variant sequencesSEQ ID Nos: 250-288), FbCas12a (variant sequences SEQ ID Nos: 289-327),Lb4Cas12a (variant sequences SEQ ID Nos: 328-366), MbCas12a (variantsequences SEQ ID Nos: 367-405), Pb2Cas12a (variant sequences SEQ ID Nos:406-444), PgCas12a (variant sequences SEQ ID Nos: 445-483), AaCas12a(variant sequences SEQ ID Nos: 484-522), BoCas12a (variant sequences SEQID Nos: 523-561), and CmaCas12a (variant sequences SEQ ID Nos: 562-600).In some embodiments, the single-strand-specific Cas12a nucleicacid-guided nuclease is at least 70% identical to any one of SEQ ID NOs:16-600 and contains an amino acid substitution(s) listed in Tables 9-13or the equivalent in a different ortholog. In some embodiments, thesingle-strand-specific Cas12a nucleic acid-guided nuclease is at least75% identical to any one of SEQ ID NOs: 16-600 and contains an aminoacid substitution(s) listed in Tables 9-13 or the equivalent in adifferent ortholog. In some embodiments, the single-strand-specificCas12a nucleic acid-guided nuclease is at least 80% identical to any oneof SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listedin Tables 9-13 or the equivalent in a different ortholog. In someembodiments, the single-strand-specific Cas12a nucleic acid-guidednuclease is at least 85% identical to any one of SEQ ID NOs: 16-600 andcontains an amino acid substitution(s) listed in Tables 9-13 or theequivalent in a different ortholog. In some embodiments, thesingle-strand-specific Cas12a nucleic acid-guided nuclease is at least90% identical to any one of SEQ ID NOs: 16-600 and contains an aminoacid substitution(s) listed in Tables 9-13 or the equivalent in adifferent ortholog. In some embodiments, the single-strand-specificCas12a nucleic acid-guided nuclease is at least 95% identical to any oneof SEQ ID NOs: 16-600 and contains an amino acid substitution(s) listedin Tables 9-13 or the equivalent in a different ortholog. In someembodiments, the single-strand-specific Cas12a nucleic acid-guidednuclease is at least %, 97%, 98% or 99% identical to any one of SEQ IDNOs: 16-600 and contains an amino acid substitution(s) listed in Tables9-13 or the equivalent in a different ortholog. In some embodiments, thesingle-strand-specific Cas12a nucleic acid-guided nuclease is any one ofSEQ ID NOs: 16-600.

The single-strand-specific Cas12a nucleic acid-guided nucleasesdescribed herein may be any Cas12a nucleic acid-guided nuclease thatlargely prevents double-stranded nucleic acid unwinding and R-loopformation. The single-strand-specific Cas12a nucleic acid-guidednucleases described herein may also be any Cas12a nucleic acid-guidednuclease that lacks cis-cleavage activity yet maintains trans-nucleicacid-guided nuclease activity on single-stranded nucleic acid molecules.Such single-strand-specific Cas12a nucleic acid-guided nucleases may begenerated via the mutations described herein.

Additionally, or alternatively, such single-strand-specific Cas12anucleic acid-guided nucleases may be generated via post-translationalmodifications (e.g., acetylation). The single-strand-specific Cas12anucleic acid-guided nucleases of the disclosure may be an acetylatedCas12a enzyme. The single-strand-specific Cas12a nucleic acid-guidednucleases of the disclosure may be an LbCas12a (i.e., SEQ ID NO: 1) withan acetylated K595 (K595K^(Ac)) residue. The single-strand-specificCas12a nucleic acid-guided nucleases of the disclosure may be anAsCas12a (i.e., SEQ ID NO: 2) with an acetylated K607 (K607K^(Ac))residue. The single-strand-specific Cas12a nucleic acid-guided nucleasesof the disclosure may be a CtCas12a (i.e., SEQ ID NO: 3) with anacetylated R591 (R591R^(Ac)) residue. The single-strand-specific Cas12anucleic acid-guided nucleases of the disclosure may be an EeCas12a(i.e., SEQ ID NO: 4) with an acetylated K601 (K607K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be an Mb3Cas12a (i.e., SEQ ID NO: 5) with an acetylatedK635 (K635K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be an FnCas12a (i.e., SEQ IDNO: 6) with an acetylated K671 (K671K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be an FnoCas12a (i.e., SEQ ID NO: 7) with an acetylatedN671 (N671K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be an FbCas12a (i.e., SEQ IDNO: 8) with an acetylated K678 (K678K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be an Lb4Cas12a (i.e., SEQ ID NO: 9) with an acetylatedK601 (K601K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be an MbCas12a (i.e., SEQ IDNO: 10) with an acetylated K625 (K625K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be a Pb2Cas12a (i.e., SEQ ID NO: 11) with an acetylatedK619 (K619K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be a PgCas12a (i.e., SEQ IDNO: 12) with an acetylated K732 (K732K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be an AaCas12a (i.e., SEQ ID NO: 13) with an acetylatedK607 (K607K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be an BoCas12a (i.e., SEQ IDNO: 14) with an acetylated K653 (K653K^(Ac)) residue. Thesingle-strand-specific Cas12a nucleic acid-guided nucleases of thedisclosure may be an CmaCas12a (i.e., SEQ ID NO: 15) with an acetylatedK577 (K577K^(Ac)) residue. The single-strand-specific Cas12a nucleicacid-guided nucleases of the disclosure may be a Cas12a orthologacetylated at the amino acid of the ortholog equivalent to K595 of SEQID NO:1. Acetylation of Cas12a can be carried out with any suitableacetyltransferase. For a discussion and methods for disabling of Cas12aby ArVA5, see Dong, et al., Nature Structural and Molecular Bio.,26(4):308-14 (2019). For example, LbCas12a can be incubated with AcrVA5in order to acetylate the K595 residue, thereby deactivating the dsDNAactivity (e.g., FIG. 7 ). In addition to acetylation, phosphorylationand methylation of select amino acid residues may be employed.

Bulky Modifications

In addition to the modalities of adjusting the ratio of theconcentration of the blocked nucleic acid molecules to the concentrationof the RNP2 and altering the domains of the variant nucleic acid-guidednuclease of RNP2 that interact with the PAM region or surroundingsequences on the blocked nucleic acid molecules to vary dsDNA vs. ssDNArecognition properties as described in detail above, the presentdisclosure additionally contemplates use of “bulky modifications” at the5′ and/or 3′ ends and/or at internal nucleic acid bases of the blockednucleic acid molecule and/or using modifications between internalnucleic acid bases. FIG. 8A is an illustration of the steric hindranceat the PAM-interacting (PI) domain in a nucleic acid-guided nucleasecaused by 5′ and 3′ modifications to a blocked nucleic acid molecule. Attop in FIG. 8A is an illustration of the target stand and non-targetstrand, and below this is an illustration of a self-hybridized blockednucleic acid molecule comprising three loop regions, as well as bulkymodifications on the 5′ and 3′ ends of the blocked nucleic acidmolecule. Example “bulky modifications” include a fluorophore andquencher pair (as shown here) or biotin, but in general encompassmolecules with a size of about 1 nm or less, or 0.9 nm or less, or 0.8nm or less, or 0.7 nm or less, or 0.6 nm or less, or 0.5 nm or less, or0.4 nm or less, or 0.3 nm or less, or 0.2 nm or less, or 0.1 nm or less,or 0.05 nm or less, or as small as 0.025 nm or less.

In the illustration at center, the blocked nucleic acid molecule withthe 5′ and 3′ ends comprising a fluorophore and a quencher is shownbeing cleaved at the loop regions. Note that the bulky modifications inthis embodiment also allow the blocked nucleic acid molecule to act as areporter moiety; that is, when the loop regions of the blocked nucleicacid molecule are cleaved, the short nucleotide segments of thenon-target strand dehybridize from the target strand due to low T_(m),thereby separating the fluorophore and quencher such that fluorescencefrom the fluorophore is no longer quenched and can be detected. In theillustration at bottom, the intact blocked nucleic acid molecule withthe bulky modifications (at left) sterically hinders interaction withthe PAM-interacting (PI) domain of the nucleic acid-guided nuclease inRNP2 such that the intact blocked nucleic acid molecule cannot becleaved via cis-cleavage by the nucleic acid-guided nuclease. However,once the loop regions of the blocked nucleic acid molecule are cleaved(via, e.g., trans-cleavage from RNP1 (at right)) and the shortnucleotide segments of the non-target strand dehybridize from the targetstrand, leaving the 3′ end of the now single-stranded target strand isnow free to initiate R-loop formation with RNP2. R-loop formation leadsto cis-cleavage of the single-strand target strand, and subsequentactivation of trans-cleavage of RNP2.

FIG. 8B illustrates five exemplary variations of blocked nucleic acidmolecules with bulky modifications, including at the 5′ and/or 3′ endsof a self-hybridizing blocked nucleic acid molecule and/or at internalnucleic acid bases of the blocked nucleic acid molecule. Embodiment (i)illustrates a self-hybridizing blocked nucleic acid molecule having afluorophore at its 5′ end and a quencher at its 3′ end. Embodiment (ii)illustrates a self-hybridizing blocked nucleic acid molecule having afluorophore and a quencher at internal nucleic acid bases flanking aloop sequence. Embodiment (iii) illustrates a self-hybridizing blockednucleic acid molecule having a fluorophore at its 5′ end and a quencherat its 3′ end as well as having a fluorophore and a quencher at internalnucleic acid bases where the internal fluorophore and quencher flank aloop sequence. The fluorophore/quencher embodiments work as long as thefluorophore and quencher are at a distance of about 10-11 nm or lessapart. Embodiment (iv) illustrates a self-hybridizing blocked nucleicacid molecule having a biotin molecule at its 5′ end, and embodiment (v)illustrates a self-hybridizing blocked nucleic acid molecule having abiotin at an internal nucleic acid base. Note that bulky modificationsof internal nucleic acid bases often are made at or near a loop regionof a blocked nucleic acid molecule (or blocked target molecule). Theloop regions are regions of the blocked nucleic acid molecules—inaddition to the 5′ and 3′ ends—that may be vulnerable to unwinding.

Modifications can be used in self-hybridized blocked nucleic acidmolecules lacking a PAM or those comprising a PAM, partiallyself-hybridized blocked nucleic acid molecules lacking a PAM or thosecomprising a PAM, or reverse PAM molecules. Other variations includeusing RNA loops instead of DNA loops if a Cas 13 nucleic acid-guidednuclease is used as the nucleic acid-guided nuclease in RNP1, or entireRNA molecules if a Cas 13 nucleic acid-guided nuclease is used as thenucleic acid-guided nuclease in RNP1 and RNP2.

FIGS. 8C, 8D and 8E list exemplary bulky modifications for 5′, 3′, andinternal positions in blocked nucleic acid molecules, and Table 14 belowlists sequences of exemplary self-hybridizing blocked nucleic acidmolecules. 56-FAM stands for 5′6-FAM (6-fluorescein amidite); and 3BHQstands for 3′ BLACK HOLE QUENCHER®-1.

TABLE 14 Bulky Modifications SEQ ID Molecule No. NO: NameMolecule Sequence (5′→3′) 5' FAM + 3' BHQ  1 601 5’F_U29_Q /56-FAM/GATCCATTTTATTTTAGATCATATATATACATGATCGG ATC/3BHQ_1/  2 602 5’F_1C/56- armor_ FAM/CGATCCATTTTATTTTAGATCATATATATACATGATCG U29_QGATCG/3BHQ_1/  3 603 5’F_2CC /56- armor_FAM/CCGATCCATTTTATTTTAGATCATATATATACATGATC U29_Q GGATCGG/3BHQ_1/  4 6045’F_1A /56- armor_ FAM/AGATCCATTTTATTTTAGATCATATATATACATGATCG U29_QGATCT/3BHQ_1/  5 605 5’F_2AT /56- armor_FAM/ATGATCCATTTTATTTTAGATCATATATATACATGATC U29_Q GGATCAT/3BHQ_1/  6 6065’F_U250_ /56- Q FAM/GATATATAAAAAAAAAAAGATCATATACATATATGATCATATATC/3BHQ_1/  7 607 5’F_1C /56- armor_FAM/CGATATATAAAAAAAAAAAGATCATATACATATATGA U250_Q TCATATATCG/3BHQ_1/  8608 5’F_2CC /56- armor_ FAM/CCGATATATAAAAAAAAAAAGATCATATACATATATG U250_QATCATATATCGG/3BHQ_1/  9 609 5’F_1A /56- armor_FAM/AGATATATAAAAAAAAAAAGATCATATACATATATGA U250_Q TCATATATCT/3BHQ_1/ 10610 5’F_2AT /56- armor_ FAM/ATGATATATAAAAAAAAAAAGATCATATACATATATG U250_QATCATATATCAT/3BHQ_1/ 5' Fluorsceine (modification on base) + 3' BHQ 11611 5’FdT_ /SFluorT/GATCCATTTTATTTTAGATCATATATATACATGATC U29_QGGATCA/3BHQ_1/ 12 612 5’FdT_1C/SFluorT/CGATCCATTTTATTTTAGATCATATATATACATGAT armor_ CGGATCGA/3BHQ_1/U29_Q 13 605 5’FdT_1A A/iFluorT/GATCCATTTTATTTTAGATCATATATATACATGATarmor_ CGGATCAT/3BHQ_1/ U29_Q 14 613 5’FdT_/SFluorT/GATATATAAAAAAAAAAAGATCATATACATATATG U250_Q ATCATATATCA/3BHQ_1/15 614 5’FdT_1C /SFluorT/CGATATATAAAAAAAAAAAGATCATATACATATAT armor_GATCATATATCGA/3BHQ_1/ U250_Q 16 610 5’FdT_1AA/iFluorT/GATATATAAAAAAAAAAAGATCATATACATATAT armor_GATCATATATCAT/3BHQ_1/ U250_Q5' FAM + Internal Fluorsceine (modification on base) + 3' BHQ 17 6015’F_IntFdt_ /56- U29_Q FAM/GA/iFluorT/CCATTTTATTTTAGATCATATATATACATGATCGGATC/3BHQ_1/ 18 606 5’F_IntFdt_ /56- U250_QFAM/GA/iFluorT/ATATAAAAAAAAAAAGATCATATACATAT ATGATCATATATC/3BHQ_1/ 19602 5’F_1C /56- armor_ FAM/CGA/iFluorT/CCATTTTATTTTAGATCATATATATACATIntFdt_U29_Q GATCGGATCG/3BHQ_1/ 20 604 5’F_1A /56- armor_FAM/AGA/iFluorT/CCATTTTATTTTAGATCATATATATACAT IntFdt_U29_QGATCGGATCT/3BHQ_1/ 21 607 5’F_1C /56- armor_FAM/CGA/iFluorT/ATATAAAAAAAAAAAGATCATATACATA IntFdt_U250_QTATGATCATATATCG/3BHQ_1/ 22 609 5’F_1A /56- armor_FAM/AGA/iFluorT/ATATAAAAAAAAAAAGATCATATACATA IntFdt_U250_QTATGATCATATATCT/3BHQ_1/ 23 603 5’F_2CC /56- armor_FAM/CCGA/iFluorT/CCATTTTATTTTAGATCATATATATACA IntFdt_U29_QTGATCGGATCGG/3BHQ_1/ 24 605 5’F_2AT /56- armor_FAM/ATGA/iFluorT/CCATTTTATTTTAGATCATATATATACA IntFdt_U29_QTGATCGGATCAT/3BHQ_1/ 25 608 5'F_2CC /56- armor_FAM/CCGA/iFluorT/ATATAAAAAAAAAAAGATCATATACAT dIntFt_U250_QATATGATCATATATCGG/3BHQ_1/ 26 610 5’F_2AT /56- armor_FAM/ATGA/iFluorT/ATATAAAAAAAAAAAGATCATATACAT IntFdt_U250_QATATGATCATATATCAT/3BHQ_1/

Applications of the Cascade Assay

The present disclosure describes cascade assays for detecting a targetnucleic acid of interest in a sample that provide instantaneous ornearly instantaneous results even at ambient temperatures at 16° C. andabove, allow for massive multiplexing and minimum workflow, yet provideaccurate results at low cost. Moreover, the various embodiments of thecascade assay are notable in that, with the exception of the gRNA inRNP1, the cascade assay components stay the same no matter what targetnucleic acid(s) of interest are being detected and RNP1 is easilyreprogrammed. Moreover, the cascade assay can be massively multiplexedfor detecting several to many to target nucleic acid moleculessimultaneously. For example, the assay may be designed to detect one toseveral to many different pathogens (e.g., testing for many differentpathogens in one assay), or the assay may be designed to detect one toseveral to many different sequences from the same pathogen (e.g., toincrease specificity and sensitivity), or a combination of the two.

As described above, early and accurate identification of, e.g.,infectious agents, microbe contamination, and variant nucleic acidsequences that indicate the present of such diseases such as cancer orcontamination by heterologous sources is important in order to selectcorrect therapeutic treatment, identify tainted food, pharmaceuticals,cosmetics and other commercial goods; and to monitor the environment.The cascade assay described herein can be applied in diagnostics for,e.g., infectious disease (including but not limited to Covid, HIV, flu,the common cold, Lyme disease, STDs, chicken pox, diptheria,mononucleosis, hepatitis, UTIs, pneumonia, tetanus, rabies, malaria,dengue fever, Ebola, plague; see Table 1), for rapid liquid biopsies andcompanion diagnostics (biomarkers for cancers, early detection,progression, monitoring; see Table 4), prenatal testing (including butnot limited to chromosomal abnormalities and genetic diseases such assickle cell, including over-the-counter versions of prenatal testingassays), rare disease testing (achondroplasia, Addison's disease,α1-antitrypsin deficiency, multiple sclerosis, muscular dystrophy,cystic fibrosis, blood factor deficiencies), SNP detection/DNAprofiling/epigenetics, genotyping, low abundance transcript detection,labeling for cell or droplet sorting, in situ nucleic acid detection,sample prep, library quantification of NGS, screening biologics(including engineered therapeutic cells for genetic integrity and/orcontamination), development of agricultural products, food compliancetesting and quality control (e.g., detection of genetically modifiedproducts, confirmation of source for high value commodities,contamination detection), infectious disease in livestock, infectiousdisease in cash crops, livestock breeding, drug screening, personalgenome testing including clinical trial stratification, personalizedmedicine, nutrigenomics, drug development and drug therapy efficacy,transplant compatibility and monitoring, environmental testing andforensics, and bioterrorism agent monitoring.

Target nucleic acids of interest are derived from samples as describedin more detail above. Suitable samples for testing include, but are notlimited to, any environmental sample, such as air, water, soil, surface,food, clinical sites and products, industrial sites and products,pharmaceuticals, medical devices, nutraceuticals, cosmetics, personalcare products, agricultural equipment and sites, and commercial samples,and any biological sample obtained from an organism or a part thereof,such as a plant, animal, or microbe. In some embodiments, the biologicalsample is obtained from an animal subject, such as a human subject. Abiological sample may be any solid or fluid sample obtained from,excreted by or secreted by any living organism, including, withoutlimitation, single celled organisms, such as bacteria, yeast,protozoans, and amoebas among others, multicellular organisms includingplants or animals, including samples from a healthy or apparentlyhealthy human subject or a human patient affected by a condition ordisease to be diagnosed or investigated, such as an infection with apathogenic microorganism, such as a pathogenic bacteria or virus.

For example, a biological sample can be a biological fluid obtained froma human or non-human (e.g., livestock, pets, wildlife) animal, and mayinclude but is not limited to blood, plasma, serum, urine, stool,sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleuraleffusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreoushumor, or any bodily secretion, a transudate, an exudate (for example,fluid obtained from an abscess or any other site of infection orinflammation), or fluid obtained from a joint (for example, a normaljoint or a joint affected by disease, such as rheumatoid arthritis,osteoarthritis, gout or septic arthritis), or a swab of skin or mucosalmembrane surface (e.g., a nasal or buccal swab).

In some embodiments, the sample can be a viral or bacterial sample or abiological sample that has been minimally processed, e.g., only treatedwith a brief lysis step prior to detection. In other embodiments,minimal processing can include thermal lysis at an elevated temperatureto release nucleic acids. Suitable methods are contemplated in U.S. Pat.No. 9,493,736, among other references. Common methods for cell lysisinvolve thermal, chemical, enzymatic, or mechanical treatment of thesample or a combination of those (see, e.g., Example I below). In someembodiments, minimal processing can include treating the sample withchaotropic salts such as guanidine isothiocyanate or guanidine HCl.Suitable methods are contemplated in U.S. Pat. Nos. 8,809,519 and7,893,251, among other references. In some embodiments, minimalprocessing may include contacting the sample with reducing agents suchas DTT or TCEP and EDTA to inactivate inhibitors and/or other nucleasespresent in the crude samples. In other embodiments, minimal processingfor biofluids may include centrifuging the samples to obtain cell-debrisfree supernatant before applying the reagents. Suitable methods arecontemplated in U.S. Pat. No. 8,809,519, among other references. Instill other embodiments, minimal processing may include performingDNA/RNA extraction to get purified nucleic acids before applying CRISPRCascade reagents.

Table 15 below lists exemplary commercial sample processing kits, andTable 16 below lists point of care processing techniques.

TABLE 15 Exemplary Commercial Sample and Nucleic Acid Processing KitsManufacturer Kit Sample Type Output Lysing and extraction methodsQiagen ® DNeasy ™ Blood small volumes genomic Isolation of Genomic DNAfrom Small & Tissue Kits of blood DNA Volumes of Blood dried blood 1.Uses Chemical and spots Biological/Enzymatic lysis methods urine 2. UsesSPE with Column Purification tissues Isolation of Genomic DNA fromTissues laser- 1. Uses Chemical and microdissected Biological/Enzymaticlysis methods tissues 2. Used to dissolve and lyse tissue sectionscompletely, higher temperature and longer time incubations up to 24hours are used Qiagen ® QIAamp ® UCP whole blood microbial Specificpretreatment protocols are Pathogen swabs DNA suggested depending onsample type with Mini Handbook cultures— or without the use of kits forMechanical microbial DNA pelleted Lysis Method before downstreampurification microbial cells applications. body fluids Downstreamapplications contain: 1. Chemical and Biological/Enzymatic lysis methods2. SPE with Column Purification Qiagen ® QIAamp ® Viral plasma and viralDNA 1. Uses Chemical lysis methods RNA Kits serum 2. Uses SPE withColumn Purification CSF urine other cell-free body fluids cell-culturesupernatants swabs Zymo Quick- whole blood genomic 1. Uses chemicallysis methods Research ™ DNA ™Microprep plasma DNA 2. Uses SPE withcolumn purification Kit serum body fluids buffy coat lymphocytes swabscultured cells Zymo Quick-DNA ™ A. fumigatus Microbial Uses Bead lysisand pretreatment with: Research ™ Fungal/Bacterial C. albicans DNA 1.Chemical lysis methods with Miniprep Kit N. crassa chaotropic salts S.cerevisiae 2. NAE with SPE with silica matrices S. pombe mycelium Grampositive bacteria Gram negative bacteria

TABLE 16 Point of Care Sample Processing Techniques Steps ProtocolExample 1 Protocol Example 2 Protocol Example 3 Field-deployable viralStreamlined Lucira Health ™ diagnostics using inactivation, CRISPR-Cas13amplification, and Science, Cas13-based detection 27; 360(6387):444-448of SARS-CoV-2 (2018) NatCommun, 11: 5921 (2020) 1. Cell disruptionSamples were thermally A NP swab or saliva Lucira Health uses a (lysis)and treated at ~40° C. for ~15 sample was lysed and single buffer thatlyses inactivation of minutes for nuclease inactivated for 10 andinactivates nucleases deactivation, thereafter minutes with thermalnucleases and/or In POC setting, cell at 90° C. for 5 minutes treatment.These inhibitors. disruption and for viral deactivation. samples wereincubated A nasal swab is directly inactivation of Sample Types: for 5min at 40° C., added to a single nucleases is done Urine followed by 5min at lysing/reaction buffer commonly through Saliva 70° C. (or 5 minat 95° C., and vigorously stirred thermal lysis. Diluted blood ifsaliva) to release the viral (1:3 with PBS) particulates from theTargets: Viruses swab. Target: SARS-Cov-2 2. Assay on crude Thermallytreated Thermally treated Processed biological sample biologicalbiological sample is used in an This is usually a direct samples(above)were samples(above) were isothermal reaction for assay on the crude useddirectly for used directly for pathogenic nucleic acid sample post cellamplification and amplification and detection. disruption and detectionof pathogenic detection of pathogenic inactivation of nucleic acid.nucleic acid. nucleases. No extraction is usually performed.

FIG. 9 shows a lateral flow assay (LFA) device that can be used todetect the cleavage and separation of a signal from a reporter moiety.For example, the reporter moiety may be a single-stranded ordouble-stranded oligonucleotide with terminal biotin and fluoresceinamidite (FAM) modifications; and, as described above, the reportermoiety may also be part of a blocked nucleic acid. The LFA device mayinclude a pad with binding particles, such as gold nanoparticlesfunctionalized with anti-FAM antibodies; a control line with a firstbinding moiety attached, such as avidin or streptavidin; a test linewith a second binding moiety attached, such as antibodies; and anabsorption pad. After completion of a cascade assay (see FIGS. 2A, 3A,and 3B), the assay reaction mix is added to the pad containing thebinding particles, (e.g., antibody labeled gold nanoparticles). When thetarget nucleic acid of interest is present, a reporter moiety iscleaved, and when the target nucleic acid of interest is absent, thereporter is not cleaved.

A moiety on the reporter binds to the binding particles and istransported to the control line. When the target nucleic acid ofinterest is absent, the reporter moiety is not cleaved, and the firstbinding moiety binds to the reporter moiety, with the binding particlesattached. When the target nucleic acid of interest is present, oneportion of the cleaved reporter moiety binds to the first bindingmoiety, and another portion of the cleaved reporter moiety bound to thebinding particles via the moiety binds to the second binding moiety. Inone example, anti-FAM gold nanoparticles bind to a FAM terminus of areporter moiety and flow sequentially toward the control line and thento the test line. For reporters that are not trans-cleaved, goldnanoparticles attach to the control line via biotin-streptavidin andresult in a dark control line. In a negative test, since the reporterhas not been cleaved, all gold conjugates are trapped on control linedue to attachment via biotin-streptavidin. A negative test will resultin a dark control line with a blank test line. In a positive test,reporter moieties have been trans-cleaved by the cascade assay, therebyseparating the biotin terminus from the FAM terminus. For cleavedreporter moieties, nanoparticles are captured at the test line due toanti-FAM antibodies. This positive test results in a dark test line inaddition to a dark control line.

The components of the cascade assay may be provided in various kits fortesting at, e.g., point of care facilities, in the field, pandemictesting sites, and the like. In one aspect, the kit for detecting atarget nucleic acid of interest in a sample includes: firstribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes(RNP2s), blocked nucleic acid molecules, and reporter moieties. Thefirst complex (RNP1) comprises a first nucleic acid-guided nuclease anda first gRNA, where the first gRNA includes a sequence complementary tothe target nucleic acid(s) of interest. Binding of the first complex(RNP1) to the target nucleic acid(s) of interest activatestrans-cleavage activity of the first nucleic acid-guided nuclease. Thesecond complex (RNP2) comprises a second nucleic acid-guided nucleaseand a second gRNA that is not complementary to the target nucleic acidof interest. The blocked nucleic acid molecule comprises a sequencecomplementary to the second gRNA, where trans-cleavage of the blockednucleic acid molecule results in an unblocked nucleic acid molecule andthe unblocked nucleic acid molecule can bind to the second complex(RNP2), thereby activating the trans-cleavage activity of the secondnucleic acid-guided nuclease. Activating trans-cleavage activity in RNP2results in an exponential increase in unblocked nucleic acid moleculesand in active reporter moieties, where reporter moieties are nucleicacid molecules and/or are operably linked to the blocked nucleic acidmolecules and produce a detectable signal upon cleavage by RNP2.

In a second aspect, the kit for detecting a target nucleic acid moleculein sample includes: first ribonucleoprotein complexes (RNP1s), secondribonucleoprotein complexes (RNP2s), template molecules, blocked primermolecules, a polymerase, NTPs, and reporter moieties. The firstribonucleoprotein complex (RNP1) comprises a first nucleic acid-guidednuclease and a first gRNA, where the first gRNA includes a sequencecomplementary to the target nucleic acid of interest and where bindingof RNP1 to the target nucleic acid(s) of interest activatestrans-cleavage activity of the first nucleic acid-guided nuclease. Thesecond complex (RNP2) comprises a second nucleic acid-guided nucleaseand a second gRNA that is not complementary to the target nucleic acidof interest. The template molecules comprise a primer binding domain(PBD) sequence as well as a sequence corresponding to a spacer sequenceof the second gRNA. The blocked primer molecules comprise a sequencethat is complementary to the PBD on the template nucleic acid moleculeand a blocking moiety.

Upon binding to the target nucleic acid of interest, RNP1 becomes activetriggering trans-cleavage activity that cuts at least one of the blockedprimer molecules to produce at least one unblocked primer molecule. Theunblocked primer molecule hybridizes to the PBD of one of the templatenucleic acid molecules, is trimmed of excess nucleotides by the 3′-to-5′exonuclease activity of the polymerase and is then extended by thepolymerase and NTPs to form a synthesized activating molecule with asequence that is complementary to the second gRNA of RNP2 (i.e., thesynthesized activating molecule is the target strand). Upon activatingRNP2, additional trans-cleavage activity is initiated, cleaving at leastone additional blocked primer molecule. Continued cleavage of blockedprimer molecules and subsequent activation of more RNP2s proceeds at anexponential rate. A signal is generated upon cleavage of a reportermolecule by active RNP2 complexes; therefore, a change in signalproduction indicates the presence of the target nucleic acid molecule.

Any of the kits described herein may further include a sample collectiondevice, e.g., a syringe, lancet, nasal swab, or buccal swab forcollecting a biological sample from a subject, and/or a samplepreparation reagent, e.g., a lysis reagent. Each component of the kitmay be in separate container or two or more components may be in thesame container. The kit may further include a lateral flow device usedfor contacting the biological sample with the reaction mixture, where asignal is generated to indicate the presence or absence of the targetnucleic acid molecule of interest. In addition, the kit may furtherinclude instructions for use and other information.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example I Preparation of Nucleic Acids of Interest

Mechanical lysis: Nucleic acids of interest may be isolated by variousmethods depending on the cell type and source (e.g., tissue, blood,saliva, environmental sample, etc.). Mechanical lysis is a widely usedcell lysis method and may be used to extract nucleic acids frombacterial, yeast, plant and mammalian cells. Cells are disrupted byagitating a cell suspension with “beads” at high speeds (beads fordisrupting various types of cells can be sourced from, e.g., OPSDiagnostics (Lebanon N.J., US) and MP Biomedicals (Irvine, Calif.,USA)). Mechanical lysis via beads begins with harvesting cells in atissue or liquid, where the cells are first centrifuged and pelleted.The supernatant is removed and replaced with a buffer containingdetergents as well as lysozyme and protease. The cell suspension ismixed to promote breakdown of the proteins in the cells and the cellsuspension then is combined with small beads (e.g., glass, steel, orceramic beads) that are mixed (e.g., vortexed) with the cell suspensionat high speeds. The beads collide with the cells, breaking open the cellmembrane with shear forces. After “bead beating”, the cell suspension iscentrifuged to pellet the cellular debris and beads, and the supernatantmay be purified via a nucleic acid binding column (such as the MagMAX™Viral/Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham,Mass., USA) and others from Qiagen (Hilden, Germany), TakaraBio (SanJose, Calif., USA), and Biocomma (Shenzen, China)) to collect thenucleic acids (see the discussion of solid phase extraction below).

Solid phase extraction (SPE): Another method for capturing nucleic acidsis through solid phase extraction. SPE involves a liquid and stationaryphase, which selectively separates the target analyte (here, nucleicacids) from the liquid in which the cells are suspended based onspecific hydrophobic, polar, and/or ionic properties of the targetanalyte in the liquid and the stationary solid matrix. Silica bindingcolumns and their derivatives are the most commonly used SPE techniques,having a high binding affinity for DNA under alkaline conditions andincreased salt concentration; thus, a highly alkaline and concentratedsalt buffer is used. The nucleic acid sample is centrifuged through acolumn with a highly porous and high surface area silica matrix, wherebinding occurs via the affinity between negatively charged nucleic acidsand positively charged silica material. The nucleic acids bind to thesilica matrices, while the other cell components and chemicals passthrough the matrix without binding. One or more wash steps typically areperformed after the initial sample binding (i.e., the nucleic acids tothe matrix), to further purify the bound nucleic acids, removing excesschemicals and cellular components non-specifically bound to the silicamatrix. Alternative versions of SPE include reverse SPE and ion exchangeSPE, and use of glass particles, cellulose matrices, and magnetic beads.

Thermal lysis: Thermal lysis involves heating a sample of mammaliancells, virions, or bacterial cells at high temperatures thereby damagingthe cellular membranes by denaturizing the membrane proteins.Denaturizing the membrane proteins results in the release ofintracellular DNA. Cells are generally heated above 90° C., however timeand temperature may vary depending on sample volume and sample type.Once lysed, typically one or more downstream methods, such as use ofnucleic acid binding columns for solid phase extraction as describedabove, are required to further purify the nucleic acids.

Physical lysis: Common physical lysis methods include sonication andosmotic shock. Sonication involves creating and rupturing of cavities orbubbles to release shockwaves, thereby disintegrating the cellularmembranes of the cells. In the sonication process, cells are added intolysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibitproteases. The cell samples are then placed in a water bath and asonication wand is placed directly into the sample solution. Sonicationtypically occurs between 20-50 kHz, causing cavities to be formedthroughout the solution as a result of the ultrasonic vibrations;subsequent reduction of pressure then causes the collapse of the cavityor bubble resulting in a large amount of mechanical energy beingreleased in the form of a shockwave that propagates through the solutionand disintegrates the cellular membrane. The duration of the sonicationpulses and number of pulses performed varies depending on cell type andthe downstream application. After sonication, the cell suspensiontypically is centrifuged to pellet the cellular debris and thesupernatant containing the nucleic acids may be further purified bysolid phase extraction as described above.

Another form of physical lysis is osmotic shock, which is most typicallyused with mammalian cells. Osmotic shock involves placing cells inDI/distilled water with no salt added. Because the salt concentration islower in the solution than in the cells, water is forced into the cellcausing the cell to burst, thereby rupturing the cellular membrane. Thesample is typically purified and extracted by techniques such as e.g.,solid phase extraction or other techniques known to those of skill inthe art.

Chemical lysis: Chemical lysis involves rupturing cellular and nuclearmembranes by disrupting the hydrophobic-hydrophilic interactions in themembrane bilayers via detergents. Salts and buffers (such as, e.g.,Tris-HCl pH 8) are used to stabilize pH during extraction, and chelatingagents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors(e.g., Proteinase K) are also added to preserve the integrity of thenucleic acids and protect against degradation. Often, chemical lysis isused with enzymatic disruption methods (see below) for lysing bacterialcell walls. In addition, detergents are used to lyse and break downcellular membranes by solubilizing the lipids and membrane proteins onthe surface of cells. The contents of the cells include, in addition tothe desired nucleic acids, inner cellular proteins and cellular debris.Enzymes and other inhibitors are added after lysis to inactivatenucleases that may degrade the nucleic acids. Proteinase K is commonlyadded after lysis, destroying DNase and RNase enzymes capable ofdegrading the nucleic acids. After treatment with enzymes, the sample iscentrifuged, pelleting cellular debris, while the nucleic acids remainin the solution. The nucleic acids may be further purified as describedabove.

Another form of chemical lysis is the widely used procedure ofphenol-chloroform extraction. Phenol-chloroform extraction involves theability for nucleic acids to remain soluble in an aqueous solution in anacidic environment, while the proteins and cellular debris can bepelleted down via centrifugation. Phenol and chloroform ensure a clearseparation of the aqueous and organic (debris) phases. For DNA, a pH of7-8 is used, and for RNA, a more acidic pH of 4.5 is used.

Enzymatic lysis: Enzymatic disruption methods are commonly combined withother lysis methods such as those described above to disrupt cellularwalls (bacteria and plants) and membranes. Enzymes such as lysozyme,lysostaphin, zymolase, and protease are often used in combination withother techniques such as physical and chemical lysis. For example, onecan use cellulase to disrupt plant cell walls, lysosomes to disruptbacterial cell walls and zymolase to disrupt yeast cell walls.

Example II RNP Formation

For RNP complex formation, 250 nM of LbCas12a nuclease protein wasincubated with 375 nM of a target specific gRNA in 1× Buffer (10 mMTris-HCl, 100 μg/mL BSA) with 2-15 mM MgCl₂ at 25° C. for 20 minutes.The total reaction volume was 2 μL. Other ratios of LbCas12a nuclease togRNAs were tested, including 1:1, 1:2 and 1:5. The incubationtemperature ranged from 16° C.-37° C., and the incubation time rangedfrom 10 minutes to 4 hours.

Example III Blocked Nucleic Acid Molecule Formation

Ramp cooling: For formation of the secondary structure of blockednucleic acid molecules, 2.5 μM of a blocked nucleic acid molecule (anyof Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6 ° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to 37° C. at 0.015 ° C./second to form the desired secondarystructure.

Snap cooling: For formation of the secondary structure of blockednucleic acid molecules, 2.5 μM of a blocked nucleic acid molecule (anyof Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6 ° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to room temperature by removing the heat source to form thedesired secondary structure.

Snap cooling on ice: For formation of the secondary structure of blockednucleic acid molecules, 2.5 μM of a blocked nucleic acid molecule (anyof Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl)with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to95° C. at 1.6 ° C./second and incubated at 95° C. for 5 minutes todehybridize any secondary structures. Thereafter, the reaction wascooled to room temperature by placing the reaction tube on ice to formthe desired secondary structure.

Example IV Reporter Moiety Formation

The reporter moieties used in the reactions herein were single-strandedDNA oligonucleotides 5-9 bases in length (e.g., with sequences of TTATT,TTTATTT, ATTAT, ATTTATTTA, AAAAA, or AAAAAAAAA) with a fluorophore and aquencher attached on the 5′ and 3′ ends, respectively. In one exampleusing a Cas12a cascade, the fluorophore was FAM-6 and the quencher wasIOWA BLACK® (Integrated DNA Technologies, Coralville, Iowa). In anotherexample using a Cas13 cascade, the reporter moieties weresingle-stranded RNA oligonucleotides 5-10 bases in length (e.g., r(U)n,r(UUAUU)n, r(A)n).

Example V Cascade Assay

Format I (final reaction mix components added at the same time): RNP1was assembled using the LbCas12a nuclease and a gRNA for the Methicillinresistant Staphylococcus aureus (MRSA) DNA according to the RNP complexformation protocol described in Example II (for this sequence, seeExample VI). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nMof the MRSA-target specific gRNA. Next, RNP2 was formed using theLbCas12a nuclease and a gRNA specific for a selected blocked nucleicacid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembledwith 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, Mass.) with 5 mM MgCl₂ at25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to aconcentration of 75 nM LbCas12a:112.5 nM gRNA. Thereafter, the finalreaction was carried out in 1× Buffer, with 500 nM of the ssDNA reportermoiety, 1× ROX dye (Thermo Fisher Scientific, Waltham, Mass.) forpassive reference, 2.5 mM MgCl₂, 4 mM NaCl, 15 nM LbCas12a:22.5 nM gRNARNP1, 20 nM LbCas12a:35 nM gRNA RNP2, and 50 nM blocked nucleic acidmolecule (any one of Formula I-IV) in a total volume of 9 μL. 1 μL ofMRSA DNA target (with samples having as low as three copies and as manyas 30000 copies—see FIGS. 6-14 ) was added to make a final volume of 10μL. The final reaction was incubated in a thermocycler at 25° C. withfluorescence measurements taken every 1 minute.

Format II (RNP1 and MRSA target pre-incubated before addition to finalreaction mix): RNP1 was assembled using the LbCas12a nuclease and a gRNAfor the MRSA DNA according to RNP formation protocol described inExample II (for this sequence, see Example VI). Briefly, 250 nM LbCas12anuclease was assembled with 375 nM of the MRSA-target specific gRNA.Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specificfor a selected blocked nucleic acid molecule (Formula I-IV) using 500 nMLbCas12a nuclease assembled with 750 nM of the blocked nucleicacid-specific gRNA incubated in 1× NEB 2.1 Buffer (New England Biolabs,Ipswich, Mass.) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Followingincubation, RNP1s were diluted to a concentration of 75 nMLbCas12a:112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1μL of MRSA DNA target and incubated at 16° C.-37° C. for up to 10minutes to activate RNP1. The final reaction was carried out in 1×Buffer, with 500 nM of the ssDNA reporter moiety, 1× ROX dye (ThermoFisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl₂,4 mM NaCl, the pre-incubated and activated RNP1, 20 nM LbCas12a:35 nMgRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of FormulaI-IV) in a total volume of 9 μL. The final reaction was incubated in athermocycler at 25° C. with fluorescence measurements taken every 1minute.

Format III (RNP1 and MRSA target pre-incubated before addition to finalreaction mix and blocked nucleic acid molecule added to final reactionmix last): RNP1 was assembled using the LbCas12a nuclease and a gRNA forthe MRSA DNA according to the RNP complex formation protocol describedin Example II (for this sequence, see Example VI). Briefly, 250 nMLbCas12a nuclease was assembled with 375 nM of the MRSA-target specificgRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNAspecific for a selected blocked nucleic acid molecule (Formula I-IV)using 500 nM LbCas12a nuclease assembled with 750 nM of the blockednucleic acid-specific gRNA incubated in 1× NEB 2.1 Buffer (New EnglandBiolabs, Ipswich, Mass.) with 5 mM MgCl₂ at 25° C. for 20-40 minutes.Following incubation, RNP1s were diluted to a concentration of 75 nMLbCas12a:112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1μL of MRSA DNA target and incubated at 16° C.-37° C. for up to 10minutes to activate RNP1. The final reaction was carried out in 1×Buffer, with 500 nM of the ssDNA reporter moiety, 1× ROX dye (ThermoFisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl₂,4 mM NaCl, the pre-incubated and activated RNP1, and 20 nM LbCas12a:35nM gRNA RNP2 in a total volume of 9 μL. Once the reaction mix was made,1 μL (50 nM) blocked nucleic acid molecule (any one of Formula I-IV) wasadded for a total volume of 10 μL. The final reaction was incubated in athermocycler at 25° C. with fluorescence measurements taken every 1minute.

Example VI Detection of MRSA and Test Reaction Conditions

To detect the presence of Methicillin resistant Staphylococcus aureus(MRSA) and determine the sensitivity of detection with the cascadeassay, titration experiments with a MRSA DNA target nucleic acid ofinterest were performed. The MRSA DNA sequence (NCBI Reference SequenceNC: 007793.1) is as follows.

SEQ ID NO: 615: ATGAAAAAGATAAAAATTGTTCCACTTATTTTAATAGTTGTAGTTGTCGGGTTTGGTATATATTTTTATG CTTCAAAAGATAAAGAAATTAATAATACTATTGATGCAATTGAAGATAAAAATTTCAAACAAGTTTATAA AGATAGCAGTTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATGACTGAACGTCCGATAAAAATATAT AATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAAC GAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGT TAAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTCCAGGAATGCAGAAAGACCAA AGCATACATATTGAAAATTTAAAATCAGAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCA ATACAGGAACAGCATATGAGATAGGCATCGTTCCAAAGAATGTATCTAAAAAAGATTATAAAGCAATCGC TAAAGAACTAAGTATTTCTGAAGACTATATCAAACAACAAATGGATCAAAATTGGGTACAAGATGATACC TTCGTTCCACTTAAAACCGTTAAAAAAATGGATGAATATTTAAGTGATTTCGCAAAAAAATTTCATCTTA CAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGGAAAAGCGACTTCACATCTATTAGGTTATGTTGG TCCCATTAACTCTGAAGAATTAAAACAAAAAGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAA AAGGGACTCGAAAAACTTTACGATAAAAAGCTCCAACATGAAGATGGCTATCGTGTCACAATCGTTGACG ATAATAGCAATACAATCGCACATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAGATATTCAACTAAC TATTGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATC CACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATGACGTCTATCCATTTATGTATG GCATGAGTAACGAAGAATATAATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGAT TACAACTTCACCAGGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACAAAACATTAGAC GATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACGTTA CAAGATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTT TGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATGAAAAAACTAGGTGTTGGTGAA GATATACCAAGTGATTATCCATTTTATAATGCTCAAATTTCAAACAAAAATTTAGATAATGAAATATTAT TAGCTGATTCAGGTTACGGACAAGGTGAAATACTGATTAACCCAGTACAGATCCTTTCAATCTATAGCGC ATTAGAAAATAATGGCAATATTAACGCACCTCACTTATTAAAAGACACGAAAAACAAAGTTTGGAAGAAA AATATTATTTCCAAAGAAAATATCAATCTATTAACTGATGGTATGCAACAAGTCGTAAATAAAACACATA AAGAAGATATTTATAGATCTTATGCAAACTTAATTGGCAAATCCGGTACTGCAGAACTCAAAATGAAACA AGGAGAAACTGGCAGACAAATTGGGTGGTTTATATCATATGATAAAGATAATCCAAACATGATGATGGCT ATTAATGTTAAAGATGTACAAGATAAAGGAATGGCTAGCTACAATGCCAAAATCTCAGGTAAAGTGTATG ATGAGCTATATGAGAACGGTAATAAAAAATACGATATAGATGAATAA

Briefly, a RNP1 was preassembled with a gRNA sequence designed to targetMRSA DNA. Specifically, RNP1 was designed to target a 20 bp region ofthe mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 616). An RNP2was preassembled with a gRNA sequence designed to target the unblockednucleic acid molecule that results from unblocking (i.e., linearizing)blocked nucleic acid molecule U29 (FIG. 10A). The reaction mix containedthe preassembled RNP1, preassembled RNP2, and a blocked nucleic acidmolecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mMNaCl.

FIG. 10A shows the structure and segment parameters of molecule U29.Note molecule U29 has a secondary structure free energy value of −5.84kcal/mol and relatively short self-hybridizing, double-stranded regionsof 5 bases and 6 bases. FIGS. 10B-10H show the results achieved fordetection of 3E4 copies, 30 copies, 3 copies and 0 copies of the mecAgene of MRSA (n=3) at 25° C. with varying concentrations of blockednucleic acid, RNP2 and reporter moiety. FIG. 10B shows the resultsachieved when 100 nM blocked nucleic acid molecules, 10 nM RNP2s and 500nM reporter moieties are used. Thus, in this experiment, the ratio ofblocked nucleic acid molecules to RNP2s is 10:1. Note first that with3E4 copies, nearly 100% of the reporters are cleaved at t=0 with asignal-to-noise ratio of 28.06 at 0 minutes, a signal-to-noise ratio of24.23 at 5 minutes, and a signal-to-noise ratio of 21.01 at 10 minutes.Additionally, the signal-to-noise ratios for detection with 30 copies ofMRSA target is 12.45 at 0 minutes, 14.07 at 5 minutes and 16.16 at 10minutes; and the signal-to-noise ratios for detection with 3 copies ofMRSA target is 1.79 at 0 minutes, 1.64 at 5 minutes and is 2.04 at 10minutes. Note the measured fluorescence at 0 copies increases onlyslightly over the 10- and 30-minutes intervals, resulting in a flatnegative. A flat negative (the results obtained over the time period for0 copies) demonstrates that there is very little non-specific orundesired signal generation in the system. Note that the negative whenthe ratio of blocked nucleic acid molecules to RNP2s is 10:1 is flatterthan those in FIGS. 10C through 10H.

FIG. 10C shows the results achieved when 50 nM blocked nucleic acidmolecules, 10 nM RNP2s and 500 nM reporter moieties are used. Thus, inthis experiment, the ratio of blocked nucleic acid molecules to RNP2s is5:1. Note first that with 3E4 copies, again nearly 100% of the reportersare cleaved at t=0 with a signal-to-noise ratio of 12.85, asignal-to-noise ratio of 10.51 at 5 minutes, and a signal-to-noise ratioof 8.18 at 10 minutes. Additionally, the signal-to-noise ratios fordetection with 30 copies of MRSA target is 5.85 at 0 minutes, 6.44 at 5minutes and 6.48 at 10 minutes; and the signal-to-noise ratios fordetection with 3 copies of MRSA target is 1.54 at 0 minutes, 1.61 at 5minutes and is 1.71 at 10 minutes. Note the measured fluorescence at 0copies increases, resulting in less of a flat negative than the 10:1ratio of blocked nucleic acid molecules to RNP2.

FIG. 10D shows the results achieved when 50 nM blocked nucleic acidmolecules, 10 nM RNP2s and 2500 nM reporter moieties are used. Thus, inthis experiment, the ratio of blocked nucleic acid molecules to RNP2s is5:1. With 3E4 copies, again nearly 100% of the reporters are cleaved att=0 with a signal-to-noise ratio of 34.92, a signal-to-noise ratio of30.62 at 5 minutes, and a signal-to-noise ratio of 25.81 at 10 minutes.Additionally, the signal-to-noise ratios for detection with 30 copies ofMRSA target is 7.97 at 0 minutes, 1.73 at 5 minutes and 10.50 at 10minutes; and the signal-to-noise ratios for detection with 3 copies ofMRSA target is 1.65 at 0 minutes, 1.73 at 5 minutes and is 1.82 at 10minutes. Note the measured fluorescence at 0 copies increases, resultingin less of a flat negative than the 10:1 ratio of blocked nucleic acidmolecules to RNP2s, but likely due to the 5× increase in theconcentration of reporter moieties; however, note also that a higherconcentration of reporter moieties allows for a higher signal-to-noiseratio for 3E4 and 30 copies of MRSA target.

FIG. 10E shows the results achieved when 100 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and 4 mMNaCl. Thus, in this experiment, the ratio of blocked nucleic acidmolecules to RNP2s is 5:1 but double the concentration of both of thesemolecules than that shown in FIGS. 10C and 10D. With 3E4 copies, againnearly 100% of the reporters are cleaved at t=0 with a signal-to-noiseratio of 11.89, a signal-to-noise ratio of 8.97 at 5 minutes, and asignal-to-noise ratio of 6.53 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is5.46 at 0 minutes, 5.85 at 5 minutes and 5.43 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target is1.58 at 0 minutes, 1.65 at 5 minutes and is 1.80 at 10 minutes. Note themeasured fluorescence at 0 copies increases, resulting in less of a flatnegative than the 10:1 ratio of blocked nucleic acid molecules to RNP2sshown in FIG. 10B. Note also that the ratio of blocked nucleic acidmolecules to RNP2s (5:1) appears to be more important than the ultimateconcentration (100 nM/20 nM) by comparison to FIG. 10D where the ratioof blocked nucleic acid molecules to RNP2s was also 5:1 however theconcentration of blocked nucleic acid molecules was 50 nM and theconcentration of RNP2 was 10 nM.

FIG. 1OF shows the results achieved when 50 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and using aconcentration of 4 mM NaCl. In this experiment the ratio of blockednucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, again nearly100% of the reporters are cleaved at t=0 with a signal-to-noise ratio of25.85, a signal-to-noise ratio of 21.36 at 5 minutes, and asignal-to-noise ratio of 16.24 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is5.28 at 0 minutes, 6.19 at 5 minutes and 7.02 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target isvery low at 0 minutes, 1.53 at 5 minutes and is 1.73 at 10 minutes. Notethe measured fluorescence at 0 copies increases, resulting in less of aflat negative than the 10:1 ratio of blocked nucleic acid molecules toRNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio forall concentrations was reduced at the 2.5:1 ratio of blocked nucleicacid molecules to RNP2s.

FIG. 10G shows the results achieved when 50 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and using aconcentration of 10 mM NaCl. Thus, in this experiment, the ratio ofblocked nucleic acid molecules to RNP2s is 2.5:1. With 3E4 copies, againnearly 100% of the reporters are cleaved at t=0 with a signal-to-noiseratio of 12.75, a signal-to-noise ratio of 7.78 at 5 minutes, and asignal-to-noise ratio of 3.66 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is6.09 at 0 minutes, 6.23 at 5 minutes and 3.58 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target isvery low at 0 minutes, 1.40 at 5 minutes and is 1.62 at 10 minutes. Notethe measured fluorescence at 0 copies increases, resulting in less of aflat negative than the 10:1 ratio of blocked nucleic acid molecules toRNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio forall concentrations was reduced substantially at the 2.5:1 ratio ofblocked nucleic acid molecules to RNP2s and that the NaCl concentrationat 10 mM vs. 4 mM (FIG. 10F) did not make much of a difference.

FIG. 10H shows the results achieved when 100 nM blocked nucleic acidmolecules, 20 nM RNP2s and 500 nM reporter moieties are used and using aconcentration of 10 mM NaCl. Thus, in this experiment, the ratio ofblocked nucleic acid molecules to RNP2s is 5:1. With 3E4 copies, againnearly 100% of the reporters are cleaved at t=0 with a signal-to-noiseratio of 77.38, a signal-to-noise ratio of 74.18 at 5 minutes, and asignal-to-noise ratio of 67.90 at 10 minutes. Additionally, thesignal-to-noise ratios for detection with 30 copies of MRSA target is5.94 at 0 minutes, 7,45 at 5 minutes and 9.73 at 10 minutes; and thesignal-to-noise ratios for detection with 3 copies of MRSA target is1.66 at 0 minutes, 2.13 at 5 minutes and is 2.38 at 10 minutes. Note themeasured fluorescence at 0 copies increases slightly, resulting in lessof a flat negative than the 10:1 ratio of blocked nucleic acid moleculesto RNP2s shown in FIG. 10B. Note also that the signal-to-noise ratio forall concentrations was increased substantially at the 5:1 ratio ofblocked nucleic acid molecules to RNP2s as compared to the 2.5:1 rationof blocked nucleic acid molecules to RNP2s. In summary, the resultsshown in FIGS. 10B-10H indicate that a 5:1 ratio of blocked nucleic acidmolecules to RNP2s or greater leads to higher signal-to-noise ratios forall concentrations of MRSA target.

Example VII Homology Modeling and Mutation Structure Analysis

The variant nucleic acid-guided nucleases presented herein weredeveloped in the following manner: For protein engineering and aminoacid substitution model predictions, a first Protein Data Bank (pdb)file with the amino acid sequence and structure information for the RNPcomprising the base nucleic acid-guided nuclease to be mutated, the gRNAand a bound dsDNA target nucleic acid was obtained. (For structuralinformation for RNPs comprising AsCas12s and LbCas12a, see, e.g.,Yamano, et al., Molecular Cell, 67:633-45 (2017).) Desired and/or randomamino acid substitutions were then “made” to the base nucleicacid-guided nuclease (LbCas12a)., the resulting structural change to thebase nucleic acid-guided nuclease due to each amino acid substitutionwas used to generate updated files for the resulting RNPs comprisingeach of the variant nucleic acid-guided nucleases using SWISS-MODEL andthe original pdf file as a reference template. SWISS-MODEL worked wellin the present case as the amino acid sequences of wildtype LbCas12a wasknown, as were the planned amino acid substitutions. The output of theupdated files for each variant nucleic acid-guided nuclease included aroot mean square deviation (RMSD) value for the structural changescompared to the RNP complex for wt LbCas12a in Angstrom units (i.e., ameasurement of the difference between the backbones of wt LbCas12a andthe variant nucleic acid-guided nuclease) and the updated pdb files ofthe variant nucleic acid-guided nucleases are further assessed at thepoint of mutations for changes in the hydrogen bonds compared to thereference original pdb file of the nuclease.

After SWISS modeling, an independent step for calculating free energywas performed using, e.g., a Flex ddG module based on the programRosetta CM to extract locally destabilizing mutations. This was used asa proxy for amino acid interference with PAM regions of the DNA toassess the probability of unwinding of the target nucleic acid. (See,e.g., Shanthirabalan, et al., Proteins: Structure, Function, andBioinformatics 86(8):853-867 (2018); and Barlow, et al., J. PhysicalChemistry B, 122(21):5389-99 (2018).)

Generally, the results of the SWISS-Model and Rosetta analysis indicatedthat stable enzyme function related to the PAM domain would require aglobal RMSD value range from 0.1 to 2.1 angstroms, and the following ΔΔGFlex Values: for stabilizing mutations ΔΔG≤−1.0 kcal/mol; for neutralmutations: −1.0 kcal/mol<ΔΔG<1.0 kcal/mol; and for destabilizingmutations: ΔΔG≥1.0 kcal/mol. Sixteen single mutations were identifiedthat, singly or in combination, met the calculated criteria. Structuralmodeling for mutations at four exemplary amino acid residues aredescribed below.

FIG. 6A shows the result of protein structure prediction using Rosettaand SWISS modeling of wildtype LbCas12a (Lachnospriaceae bacteriumCas12a). Protein structure prediction using Rossetta and SWISS modelingof exemplary variants of wildtype LbCas12a are shown below.

Mutation 1, G532A: The structure of an RNP comprising the G532A variantnucleic acid-guided nuclease is shown in FIG. 11A. Modeling indicatedthe following changes to the wildtype LbCas12a structure with the G532Asubstitution (seen in FIG. 11A as a red residue): loss of one hydrogenbond with TS-PAM (target strand PAM) at amino acid residue 595; loss ofone hydrogen bond with NTS-PAM (non-target strand PAM) at amino acidresidue 595; no addition or loss of a hydrogen bond at amino acidresidue 532. Per simulations, mutation G532A is a structurallystabilizing mutation. The parameters collected from SWISS-MODEL andRosetta analysis are shown in Table 17.

TABLE 17 Mutation 1: G532A Global RMSD: 0.976 PIRMSD: 0.361 REC1 RMSD:0.289 (235 to 235 atoms) WED RMSD: 0.306 (198 to 198 atoms) ΔΔG FlexValue: −1.13 PI = PAM-interacting domain of the G532A variant REC1 =REC1 domain of the G532A variant WED = WED domain of the G532A variant

Mutation 2, K538A: The structure of an RNP comprising the K538A variantnucleic acid-guided nuclease is shown at left in FIG. 11B. Modelingindicated the following changes to the wildtype LbCas12a structure withthe K538A substitution (seen in FIG. 11B as a pink residue): loss of onehydrogen bond with TS-PAM (target strand PAM) at amino acid residue 538;loss of one hydrogen bond with TS-PAM (target strand PAM) at amino acidresidue 595; loss of one hydrogen bond with NTS-PAM (non-target strandPAM) at amino acid residue 595. Per simulations, mutation K538A is astructurally stabilizing mutation. The parameters collected fromSWISS-MODEL and Rosetta analysis are shown in Table 18.

TABLE 18 Mutation 2: K538A Global RMSD: 0.990 PI RMSD: 0.376 REC1 RMSD:0.305 (236 to 236 atoms) WED RMSD: 0.324 (194 to 194 atoms) ΔΔG FlexValue: 0.06 PI = PAM-interacting domain of the K538A variant REC1 = REC1domain of the K538A variant WED = WED domain of the K538A variant

Mutation 3, Y542A: The structure of an RNP comprising the Y542A variantnucleic acid-guided nuclease is shown in FIG. 11C. Modeling indicatedthe following changes to the wildtype LbCas12a structure with the Y542Asubstitution (seen in FIG. 11C as a blue residue): loss of two hydrogenbonds with TS-PAM (target strand PAM) at amino acid residue 542; loss ofone hydrogen bond with TS-PAM (target strand PAM) at amino acid residue538; loss of one hydrogen bond with TS-PAM (target strand PAM) at aminoacid residue 595; loss of one hydrogen bond with NTS-PAM (non-targetstrand PAM) at amino acid residue 595. Per simulations, mutation Y542Ais a structurally stabilizing mutation. The parameters collected fromSWISS-MODEL and Rosetta analysis are shown in Table 19.

TABLE 19 Mutation 3: Y542A Global RMSD: 0.989 PI RMSD: 0.377 REC1 RMSD:0.306 (237 to 237 atoms) WED RMSD: 0.338 (199 to 199 atoms) ΔΔG FlexValue: −2.06 PI = PAM-interacting domain of the Y542A variant REC1 =REC1 domain of the Y542A variant WED = WED domain of the Y542A variant

Mutation 4, K595A: The structure of an RNP comprising the K595A variantnucleic acid-guided nuclease is shown in FIG. 11D. Modeling indicatedthe following changes to the wildtype LbCas12a structure with the K595Asubstitution (seen in FIG. 11D as an orange residue): loss of twohydrogen bonds with TS-PAM (target strand PAM) at amino acid residue595; loss of one hydrogen bond with NTS-PAM (non-target strand PAM) atamino acid residue 595; loss of one hydrogen bond with NTS-PAM(non-target strand PAM) at amino acid residue 538. Per simulations,mutation K595A is a structurally destabilizing mutation. The parameterscollected from SWISS-MODEL and Rosetta analysis are shown in Table 20.

TABLE 20 Mutation 4: K595A Global RMSD: 0.976 PI RMSD: 0.361 REC1 RMSD:0.289 (235 to 235 atoms) WED RMSD: 0.306 (198 to 198 atoms) ΔΔG FlexValue: 1.26 PI = PAM-interacting domain of the K595A variant REC1 = REC1domain of the K595A variant WED = WED domain of the K595A variant

Mutation 5, Combination G532A, K538A, Y542A, and K595A: The structure ofan RNP comprising the combination G532A/K538A/Y542A/K595A variant(“combination variant”) nucleic acid-guided nuclease is shown in FIG.11E. Modeling indicated the following changes to the wildtype LbCas12astructure with the four substitutions: loss of five hydrogen bonds withTS-PAM (target strand PAM); loss of one hydrogen bond with NTS-PAM(non-target strand PAM). Per simulations, the combination variant isstructurally stable. The parameters collected from SWISS-MODEL andRosetta analysis are shown in Table 21.

TABLE 21 Mutation 5: G532A/K538A/Y542A/K595A Global RMSD: 0.966 PI RMSD:0.351 REC1 RMSD: 0.261 (226 to 226 atoms) WED RMSD: 0.288 (200 to 200atoms) ΔΔG Flex Value: −3.31 PI = PAM-interacting domain of thecombination variant REC1 = REC1 domain of the combination variant WED =WED domain of the combination variant

Mutation 6, K595D: The structure of an RNP comprising the K595D variantnucleic acid-guided nuclease is shown in FIG. 11F. Modeling indicatedthe following changes to the wildtype LbCas12a structure at location 595with this substitution: loss of two hydrogen bonds with TS-PAM (targetstrand PAM); loss of one hydrogen bond with NTS-PAM (non-target strandPAM); and gain of one hydrogen bond with NTS-PAM. Per simulations, theK595D variant is structurally unstable. The parameters collected fromSWISS-MODEL and Rosetta analysis are shown in Table 22.

TABLE 22 Mutation 6: K595D Global RMSD: 1.001 PI RMSD: 0.367 (89 to 89atoms) REC1 RMSD: 0.296 (235 to 235 atoms) WED RMSD: 0.320 (197 to 197atoms) ΔΔG Flex Value: 2.04 PI = PAM-interacting domain of thecombination variant REC1 = REC1 domain of the combination variant WED =WED domain of the combination variant

Mutation 7, K595E: The structure of an RNP comprising the K595E variantnucleic acid-guided nuclease is shown in FIG. 11G. Modeling indicatedthe following changes to the wildtype LbCas12a structure at location 595with this substitution: loss of two hydrogen bonds with TS-PAM (targetstrand PAM); loss of one hydrogen bond with NTS; and no gain of hydrogenbonds. Per simulations, the K595E variant is structurally unstable. Theparameters collected from SWISS-MODEL and Rosetta analysis are shown inTable 23.

TABLE 23 Mutation 6: K595E Global RMSD: 0.975 PI RMSD: 0.352 (89 to 89atoms) REC1 RMSD: 0.264 (226 to 226 atoms) WED RMSD: 0.290 (198 to 198atoms) ΔΔG Flex Value: 1.37 PI = PAM-interacting domain of thecombination variant REC1 = REC1 domain of the combination variant WED =WED domain of the combination variant

Mutation 8, Combination K538A, Y542A, K595D: The structure of an RNPcomprising the combination K538A/Y542A/K595D variant (“combinationvariant”) nucleic acid-guided nuclease is shown in FIG. 11H. Modelingindicated the following changes to the wildtype LbCas12a structure withthe three substitutions: loss of two hydrogen bonds with TS (targetstrand) at position 595; loss of one hydrogen bond with NTS(non-target); combined loss of three hydrogen bonds at 532/242positions; and gain of one hydrogen bond at 595. Per simulations, thecombination variant is structurally destabilizing. The parameterscollected from SWISS-MODEL and Rosetta analysis are shown in Table 24.

TABLE 24 Mutation 6: K538A, Y542A, K595D Global RMSD: 0.976 PI RMSD:0.351 (89 to 89 atoms) REC1 RMSD: 0.261 (225 to 225 atoms) WED RMSD:0.289 (198 to 198 atoms) ΔΔG Flex Value: 0.96 PI = PAM-interactingdomain of the combination variant REC1 = REC1 domain of the combinationvariant WED = WED domain of the combination variant

Mutation 9, Combination K538A, Y542A, K595E: The structure of an RNPcomprising the combination K538A/Y542A/K595E variant (“combinationvariant”) nucleic acid-guided nuclease is shown in FIG. 11I. Modelingindicated the following changes to the wildtype LbCas12a structure withthe three substitutions: loss of two hydrogen bonds with TS (targetstrand) at position 595; loss of one hydrogen bond with NTS(non-target); combined loss of three hydrogen bonds at 532/242positions. Per simulations, the combination variant is structurallystabilizing. The parameters collected from SWISS-MODEL and Rosettaanalysis are shown in Table 25.

TABLE 25 Mutation 6: K538A, Y542A, K595E Global RMSD: 0.976 PI RMSD:0.351 (89 to 89 atoms) REC1 RMSD: 0.261 (225 to 225 atoms) WED RMSD:0.289 (198 to 198 atoms) ΔΔG Flex Value: −3.71 PI = PAM-interactingdomain of the combination variant REC1 = REC1 domain of the combinationvariant WED = WED domain of the combination variant

In addition to amino acid substitutions, modifications, such as chemicalmodifications, can be made to amino acids identified by the structuraland homology modeling described above. FIG. 6G illustrates an exemplaryscheme for acetylating amino acid residue 595 in LbCas12a, amodification which prevents unwinding of dsDNA by blocking entry of atarget nucleic acid into the RNP via steric hindrance. LbCas12a iscombined with AcrVA5 and the reaction is incubated for 20 minutes atroom temperature, resulting in LECas12a that has been acetylated atamino acid residue 595 (K595K^(AC)). (For a discussion and methods fordisabling of Cas12a by ArVA5, see Dong, et al., Nature Structural andMolecular Bio., 26(4):308-14 (2019).) DsDNA is not a substrate forLbCas12a with a K595K^(AC) modification; however, ssDNA is a substratefor LbCas12a with a K595K^(AC) modification; thus, LbCas12a (K595K^(AC))has the desired properties of the variant nucleic acid-guided nucleasesdescribed above. In addition to acetylation, phosphorylation andmethylation of select amino acid residues may be employed.

Example VIII Single-Strand Specificity of the Variant NucleicAcid-Guided Nucleases

In vitro transcription/translation reactions were performed for variantLbaCas12a nucleases as noted in Table 26 using the nucleic acidsequences listed in Table 27:

TABLE 26 Template DNA for IVTT 250 ng gRNA concentration 100 nMDNA activator concentration  25 nM Probe concentration 500 nMReaction volume  30 pL Reporter 5′-FAM-TTATTATT-IABkFQ-3′ PlatePCR plate 96-well, black Read temperature 25° C. Read duration30 minutes Buffer NEB r2.1 New England Biolabs ®, Inc., Ipswich, MA) Na+ 50 mM Mg + 2  10 mM

TABLE 27 Activator RunX fragment GCCTTCAGAAGAGGGTGCATTTTCAGGAGGAAGCGAT(dsDNA + PAM) GGCTTCAGACAGCATATTTGAGTCATT (SEQ ID NO. 617) RunX fragmentGCCTTCAGAAGAGGGTGCATGCACAGGAGGAAGCGAT (dsDNA - PAM)GGCTTCAGACAGCATATTTGAGTCATT (SEQ ID NO. 618) Target region inAGGAGGAAGCGATGGCTTCAGA (SEQ ID NO. 619) activator gRNA LbaCas12a gRNAgUAAUUUCUACUAAGUGUAGAUAGGAGGAAGCGAUG GCUUCAGA (SEQ ID NO. 620)The results are shown in FIGS. 12A-12G indicating the time for detectionof dsDNA and ssDNA both with and without PAM sequences for purifiedwildtype LbaCas12a and three variants (K538A+K595A, K595A, andK538A+Y542+K595A, and unpurified engineered variants ofLbaCas12a:K538D+Y542A+K595D, K595D, K538A+K595D, K538A+K595E,G532A+K538A+Y542A+K595A, K538A+Y542A+K595D, K538D+Y542A+K595A,K538D+Y542D+K595A, and K538E+Y542A+K595A. Note that all variantengineered nucleic acid-guided nucleases slowed down double-strand DNAdetection to varying degrees, with the double and triple variants atpositions K538, Y542 and K595 of wt LbaCas12a performing best incomparison to wt LbCas12a, while single-strand DNA detection remainedhigh, both in single-strand DNA with a PAM and without a PAM. Thefollowing variants were particularly robust: K538D+Y542A+K595D,K538A+K595D, K538A+K595E, G532A+K538A+Y542A+K595A, K538D+Y542A+K595A,and K538D+Y542D+K595D.

FIGS. 13A and 13B show the sequence alignment of many different Cas12anucleases and orthologs, including in some instances several alignmentsof the same Cas12a nuclease.

Example IX: Detection of Biomarker Alpha-Synuclein in CSF for MonitoringProgression of Parkinson's Disease

The biomarker α-synuclein, which is found in both aggregated andfibrillar form, has attracted attention as a biomarker of Parkinson'sdisease. Human α-synuclein is expressed in the brain in the neocortex,hippocampus, substantia nigra, thalamus and cerebellum. It is encoded bythe SNCA gene that consists of six exons ranging in size from 42 to 1110base pairs. The predominant form of α-synuclein is the full-lengthprotein, but other shorter isoforms exist. C-terminal truncation ofα-synuclein induces aggregation, suggesting that C-terminalmodifications may be involved in Parkinson's pathology. Changes in thelevels of α-synuclein have been reported in CSF of Parkinson' patients.The gradual spread of α-synuclein pathology leads to a highconcentration of extracellular α-synuclein that can potentially damagehealthy neurons. Here, the cascade assay is used to monitor the level ofnucleic acids in cerebrospinal fluid (CSF) to monitor the levels of mRNAtranscripts that when translated lead to a truncated α-synucleinprotein.

A lumbar puncture is performed on an individual, withdrawingapproximately 5 mL of cerebrospinal fluid (CSF) for testing. The CSFsample is then treated by phenol-chloroform extraction or oligo dTaffinity resins via a commercial kit (see, e.g., the TurboCapture mRNAkit or RNeaxy Pure mRNA Bead Kit from Qiagen®). Briefly, two RNP1s arepreassembled as described above in Example II with a first gRNA sequencedesigned to target the coding sequence of the mRNA transcribed from SNCAgene specific to the C-terminus region of a-synuclein to detectfull-length α-synuclein and second gRNA sequence designed to target thecoding sequence of the mRNA transcribed from SNCA gene specific to theN-terminus region of α-synuclein to detect all α-synuclein mRNAs. Inaddition to the gRNA, each RNP1 also comprises an LbCas13a nuclease(i.e., an RNA-specific nuclease). Also as described in Example II above,an RNP2 is preassembled with a gRNA sequence designed to target anunblocked nucleic acid molecule that results from unblocking (i.e.,linearizing) a chosen blocked nucleic acid molecule such as U29. Theblocked nucleic acid molecule is formed as described above in ExampleIII, and a reporter is formed as described above in Example IV. Thereaction mix contains the preassembled RNP1, preassembled RNP2, and ablocked nucleic acid molecule, in a buffer (pH of about 8) containing 4mM MgCl₂ and 101 mM NaCl. The cascade assay is performed by one of theprotocols described above in Example V. A readout is performed bycomparing the level of N-terminus coding sequences detected (the levelof total α-synuclein mRNA) versus the level of C-terminus codingsequences detected (the level of full-length α-synuclein mRNA).

Example X Detection of Foot and Mouth Disease Virus from Nasal Swabs

Foot-and-mouth disease (FMD) is a severe and highly contagious viraldisease. The FMD virus causes illness in cows, pigs, sheep, goats, deer,and other animals with divided hooves and is a worldwide concern as itcan spread quickly and cause significant economic losses. FMD hasserious impacts on the livestock trade—a single detection of FMD willstop international trade completely for a period of time. Since thedisease can spread widely and rapidly and has grave economicconsequences, FMD is one of the animal diseases livestock owners dreadmost. FMD is caused by a virus, which survives in living tissue and inthe breath, saliva, urine, and other excretions of infected animals. FMDcan also survive in contaminated materials and the environment forseveral months under the right conditions.

A nasal swab is performed on a subject, such as a cow or pig, and thenucleic acids extracted using, e.g., the Monarch Total RNA Miniprep Kit(New England Biolabs®, Inc., Ipswich, Mass.). Briefly, an RNP1 ispreassembled as described above in Example II with a gRNA sequencedesigned to a gene from the FMD virus (e.g., to a portion of NCBIReference Sequence NC 039210.1) and an LbCas12a nuclease (i.e., aDNA-specific nuclease). Also as described in Example II above, an RNP2is preassembled with a gRNA sequence designed to target an unblockednucleic acid molecule that results from unblocking (i.e., linearizing) achosen blocked nucleic acid molecule such as U29. The blocked nucleicacid molecule is formed as described above in Example III, and areporter is formed as described above in Example IV. The reaction mixcontains the preassembled RNP1, preassembled RNP2, and a blocked nucleicacid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101mM NaCl. The cascade assay is performed by one of the protocolsdescribed above in Example V, and the readout is positive detection ofFMD virus-specific DNA sequences.

Example XI Detection of Sickle Cell Gene Sequences in Peripheral Blood

Sickle cell disease (SCD) is a group of inherited red blood celldisorders. In someone who has SCD, the hemoglobin is abnormal, whichcauses the red blood cells to become hard and sticky and look like aC-shaped farm tool called a “sickle.” The sickle cells die early, whichcauses a constant shortage of red blood cells; in addition, when thesickle-shaped blood cells travel through small blood vessels, they getstuck and clog the blood flow, causing pain and other seriouscomplications such as infection and stroke.

One form of SCD is HbSS. Individuals who have this form of SCD inherittwo genes, one from each parent, that code for hemoglobin “S.”Hemoglobin S is an abnormal form of hemoglobin that causes the red cellsto become rigid and sickle shaped. This is commonly called sickle cellanemia and is usually the most severe form of the disease. Another formof SCD is HbSC. Individuals who have this form of SCD inherit ahemoglobin “S” gene from one parent and a gene for a different type ofabnormal hemoglobin called “C” from the other parent. This is usually amilder form of SCD. A third form of SCD is HbS thalassemia. Individualswho have this form of SCD inherit a hemoglobin “S” gene from one parentand a gene for beta thalassemia, another type of hemoglobin abnormality,from the other parent. There are two types of beta thalassemia: “zero”(HbS beta0) and “plus” (HbS beta+). Those with HbS beta0-thalassemiausually have a severe form of SCD. People with HbS beta+-thalassemiatend to have a milder form of SCD.

A non-invasive prenatal test (NIPT) that uses only maternal cell-freeDNA (cfDNA) from peripheral blood permits prenatal detection of sicklecell disease and beta thalassemia by screening without the need forpaternal DNA. Such a screening enables patients and healthcare providersto make informed decisions about diagnostic testing and may expand genetherapy treatment options. A 10 mL peripheral blood draw is performed ona pregnant subject into a Streck tube. The blood is treated withlysis-binding buffer and proteinase K under denaturing conditions at 55°C. for 15 minutes in the presence of magnetic beads. Following theheating step, the mixture is incubated for 1 hour at room temperaturewith mixing every 10 minutes at 1200 rpm for 30 seconds on an Eppendorfthemomixer. The beads are captured on a magnetic stand for 2 minutes,washed three times after which cfDNA is eluted by adding elution bufferand incubating for 5 minutes at 55° C. The cfDNA is further purified bydiluting in 1:1 FTA (Fast Technology for Analysis) reagent, cat#WHAWB120204 (Sigma-Aldrich, USA), containing NaCl (sodium chloride);Tris; EDTA (ethylenediaminetetraacetic acid); TRITON-X-100(t-Octylphenoxypolyethoxyethanol) and incubated for 10 minutes at roomtemperature. An additional bead purification step is performed usingPCRClean DX beads, cat #C-1003-450 (ALINE Biosciences, USA).Alternatively, there are several kits available commercially that aredesigned to extract cfDNA including the BioChain® cfPure® Cell free DNAExtraction Kit (BioChain®, Newark, Calif.); the Monarch Genomic DNAPurification Kit and the Monarch HMW DNA Extraction Kit for Blood (NewEngland Biolabs®, Inc., Ipswich, Mass.); and the cfDNA Purification Kit(Active Motif®, Carlsbad, Calif.).

For the cascade assay, three RNP1s are preassembled as described abovein Example II with 1) gRNA sequence designed to detect the Hemoglobin Sgene variant and an LbCas12a nuclease (i.e., an DNA-specific nuclease);2) a gRNA sequence designed to detect the Hemoglobin C gene variant andan LbCas 12a nuclease (i.e., an DNA-specific nuclease); and 3) a gRNAsequence designed to detect the gene for beta thalassemia and anLbCas12a nuclease (i.e., an DNA-specific nuclease). Also as described inExample II above, an RNP2 is preassembled with a gRNA sequence designedto target an unblocked nucleic acid molecule that results fromunblocking (i.e., linearizing) a chosen blocked nucleic acid moleculesuch as U29. The blocked nucleic acid molecule is formed as describedabove in Example III, and a reporter is formed as described above inExample IV. The reaction mix contains the preassembled RNP1,preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pHof about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay isperformed by one of the protocols described above in Example V. Thereadout is detection of the Hemoglobin S gene variant, the detection ofthe Hemoglobin S variant and the Hemoglobin C variant, and the detectionof the Hemoglobin S variant and the β-thalassemia gene.

Example XII Detection of Donor-Derived Gene Sequences in PeripheralBlood of Transplant Patients

Costly and invasive tissue biopsies to detect allograft rejection aftertransplantation have numerous limitations; however, assays based oncell-free DNA (cfDNA)—circulating fragments of DNA released from cells,tissues, and organs as they undergo natural cell death—can improve theability to detect rejection and implement earlier changes in managementof the transplanted organ. Rejection, referring to injury of a donatedorgan caused by the recipient's immune system, often causes allograftdysfunction and even patient death. T-cell mediated acute cellularrejection occurs most often within the first 6 months post-transplant.Acute cellular rejection involves accumulation of CD4+ and CD8+ T-cellsin the interstitial space of the allograft as the recipient's immunesystem recognizes antigens on the donated organ as foreign, initiatingan immune cascade that ultimately leads to apoptosis of the targetedcells. As these cells die, genomic DNA is cleaved and fragments of donorderived-cfDNA are released to join the pool of recipient cfDNA in theblood. Using cfDNA as a biomarker for acute cellular rejection isadvantageous since it is derived from the injured cells of the donatedorgan and therefore should represent a direct measure of cell deathoccurring in the allograft. Further, cfDNA maintains all of the geneticfeatures of the original genomic DNA, allowing the genetic materialreleased from the donated organ to be differentiated from the cfDNAderived from cells of the recipient that are undergoing naturalapoptosis.

For organ transplants in which the donor is male and the recipient isfemale, this “sex mismatch” is leveraged to calculate donorderived-cfDNA levels from within the recipient's total cfDNA pool.Although this approach allows for confident diagnosis of rejection inthe allograft, sex-mismatch between the donor and recipient isrelatively infrequent and not universally applicable; thus, the presenceof other genetic differences between the donor and recipient at aparticular locus are leveraged to identify the origin of the circulatingcfDNA. Ideally, the recipient would be homozygous for a single base (forexample, AA) and at the same locus the donor would be homozygous for adifferent base (for example, GG). Given the genetic heterogeneitybetween individuals, hundreds to tens of thousands of potentiallyinformative loci across the genome can be interrogated to distinguishdonor derived-cfDNA from recipient cfDNA.

A 10 mL peripheral blood draw is performed on a transplantation subjectinto a Streck tube. The blood is treated with lysis-binding buffer andproteinase K under denaturing conditions at 55° C. for 15 minutes in thepresence of magnetic beads. Following the heating step, the mixture isincubated for 1 hour at room temperature with mixing every 10 minutes at1200 rpm for 30 seconds on an Eppendorf themomixer. The beads arecaptured on a magnetic stand for 2 minutes, washed three times afterwhich cfDNA is eluted by adding elution buffer and incubating for 5minutes at 55° C. The cfDNA is further purified by diluting in 1:1 FTA(Fast Technology for Analysis) reagent, cat #WHAWB120204 (Sigma-Aldrich,USA), containing NaCl (sodium chloride); Tris; EDTA(ethylenediaminetetraacetic acid); TRITON-X-100(t-Octylphenoxypolyethoxyethanol) and incubated for 10 minutes at roomtemperature. An additional bead purification step is performed usingPCRClean DX beads, cat #C-1003-450 (ALINE Biosciences, USA). Also, asstated above, there are several kits available commercially that aredesigned to extract cfDNA including the BioChain® cfPure® Cell free DNAExtraction Kit (BioChain®, Newark, Calif.); the Monarch Genomic DNAPurification Kit and the Monarch HMW DNA Extraction Kit for Blood (NewEngland Biolabs®, Inc., Ipswich, Mass.); and the cfDNA Purification Kit(Active Motif®, Carlsbad, Calif.).

For the cascade assay, several to many different RNP 1 s arepreassembled as described above in Example II with gRNA sequencesdesigned to 1) query Y and/or X chromosome loci in sex mismatchtransplantation cases; or 2) gRNA sequences designed to query variousloci that are different in the genomic DNA of the recipient and thedonor; along with an LbCas12a nuclease (i.e., an DNA-specific nuclease).Also as described in Example II above, an RNP2 is preassembled with agRNA sequence designed to target an unblocked nucleic acid molecule thatresults from unblocking (i.e., linearizing) a chosen blocked nucleicacid molecule such as U29. The blocked nucleic acid molecule is formedas described above in Example III, and a reporter is formed as describedabove in Example IV. The reaction mix contains the preassembled RNP1,preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pHof about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay isperformed by one of the protocols described above in Example V. Thereadout detects the level of donor-specific nucleic acid sequences.

Example XIII Detection of Microbe Contamination in a Laboratory

DNA that is found in the environment is called “environmental DNA” oreDNA (e-DNA) for short, and it is formally defined as “genetic materialobtained directly from environmental samples without any obvious signsof biological source material.” eDNA has been harnessed to detect rareor invasive species and pathogens in a broad range of environments.Samples are typically collected in the form of water, soil, sediment, orsurface swabs. The DNA must then be extracted and purified to removechemicals that may inhibit the cascade reaction. Surface wipe samplesare commonly collected to assess microbe contamination in, e.g., alaboratory. The wipe test protocol consists of four distinct stages:removal of DNA from surfaces using absorbent wipes, extraction of DNAfrom the wipes into a buffer solution, purification of DNA, and analysisof the extract.

For sample collection, sterile 2×2 inch polyester-rayon non-woven wipesare used to wipe down an environmental surface, such as a laboratorybench. Each wipe is placed into a sterile 50 ml conical tube and 10 mLof PBST is transferred to each conical tube using a sterile serologicalpipette. The tubes are vortexed at the maximum speed for 20 minutesusing a Vortex Genie 2. A 200 μL aliquot of the supernatant wasprocessed using a nucleic acid purification kit (QIAmp DNA Blood MiniKit, QIAGEN, Inc., Valencia, Calif.). The kit lyses the sample,stabilizes and binds DNA to a selective membrane, and elutes the DNAsample.

For the cascade assay, several to many different RNP1s are preassembledas described above in Example II with gRNA sequences designed to detect,e.g., Aspergillus acidus; Parafilaria bovicola; Babesia divergens;Escherichia coli; Pseudomonas aeruginosa; and Dengue virus; along withan LbCas12a nuclease (i.e., an DNA-specific nuclease). Also as describedin Example II above, an RNP2 is preassembled with a gRNA sequencedesigned to target an unblocked nucleic acid molecule that results fromunblocking (i.e., linearizing) a chosen blocked nucleic acid moleculesuch as U29. The blocked nucleic acid molecule is formed as describedabove in Example III, and a reporter is formed as described above inExample IV. The reaction mix contains the preassembled RNP1,preassembled RNP2, and a blocked nucleic acid molecule, in a buffer (pHof about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The cascade assay isperformed by one of the protocols described above in Example V. Thereadout is detection of a genomic sequence unique to a pathogen.

While certain embodiments have been described, these embodiments havebeen presented by way of example only and are not intended to limit thescope of the present disclosures. Indeed, the novel methods,apparatuses, modules, instruments and systems described herein can beembodied in a variety of other forms; furthermore, various omissions,substitutions and changes in the form of the methods, apparatuses,modules, instruments and systems described herein can be made withoutdeparting from the spirit of the present disclosures. The accompanyingclaims and their equivalents are intended to cover such forms ormodifications as would fall within the scope and spirit of the presentdisclosures.

We claim:
 1. A method for identifying a target nucleic acid of interestin a sample in one minute or less at 16° C. comprising the steps of:providing a reaction mixture comprising: first ribonucleoprotein (RNP1)complexes (RNP1s) each comprising a first nucleic acid-guided nucleaseand a first gRNA, wherein the first gRNA comprises a sequencecomplementary to the target nucleic acid of interest; and whereinbinding of the RNP1 complex to the target nucleic acid of interestactivates cis-cleavage and trans-cleavage activity of the first nucleicacid-guided nuclease; second ribonucleoprotein complexes (RNP2s)comprising a second nucleic acid-guided nuclease and a second gRNA thatis not complementary to the target nucleic acid of interest; wherein thesecond nucleic acid-guided nuclease optionally comprises a variantnuclease engineered such that single stranded DNA is cleaved faster thandouble stranded DNA is cleaved, wherein the variant nuclease comprisesat least one mutation to the domains that interact with the PAM regionor surrounding sequences on the blocked nucleic acid molecules, andwherein the variant nuclease exhibits both cis- and trans-cleavageactivity; a plurality of blocked nucleic acid molecules comprising asequence corresponding to the second gRNA, wherein the blocked nucleicacid molecules comprise: a first region recognized by the RNP2 complex;one or more second regions not complementary to the first region formingat least one loop; one or more third regions complementary to andhybridized to the first region forming at least one clamp, whereinoptionally the molar ratio of the blocked nucleic acid molecules is atleast equal to the molar ratio of the second ribonucleoproteincomplexes, and wherein optionally the blocked nucleic acid moleculeseach comprise at least one bulky modification; and wherein one of thefollowing conditions is met: 1) providing blocked nucleic acid moleculesand ribonucleoprotein complexes where the molar ratio of the blockednucleic acid molecules is equal to or exceeds the molar ratio of theribonucleoprotein complexes, 2) the blocked nucleic acid molecules eachcomprise at least one bulky modification, or 3) the RNP2 comprises atleast one variant nuclease engineered such that single stranded DNA iscleaved faster than double stranded DNA is cleaved; and contacting thereaction mixture with the sample under conditions that allow the targetnucleic acid of interest in the sample to bind to RNP1; wherein uponbinding of the target nucleic acid of interest RNP1 becomes activeinitiating trans-cleavage of at least one of the plurality of blockednucleic acid molecules thereby producing at least one unblocked nucleicacid molecule, wherein the at least one unblocked nucleic acid moleculebinds to RNP2 initiating trans-cleavage of at least one further blockednucleic acid molecule; and detecting the cleavage products, therebydetecting the target nucleic acid of interest in the sample in oneminute or less.
 2. The method of claim 1, wherein the reaction mixturefurther comprises reporter moieties, wherein the reporter moietiesproduce a detectable signal upon trans-cleavage activity by the RNP2 toidentify the presence of one or more nucleic acid targets of interest inthe sample.
 3. The method of claim 2, wherein the reporter moieties arenot coupled to the blocked nucleic acid molecules, and wherein uponcleavage by RNP2, a signal from the reporter moiety is detected.
 4. Themethod of claim 2, wherein the reporter moieties are coupled to theblocked nucleic acid molecules, and wherein upon cleavage by RNP2, asignal from the reporter moiety is detected.
 5. The method of claim 1,wherein the reaction mixture comprises blocked nucleic acid moleculeswith bulky modifications and wherein the bulky modifications are about 1nm in size.
 6. The method of claim 6, wherein the reaction mixturecomprises blocked nucleic acid molecules with bulky modifications andwherein the bulky modifications are about 0.7 nm in size.
 7. The methodof claim 1, wherein blocked nucleic acid molecules include bulkymodifications and wherein there are two bulky modifications with onebulky modification located on the 5′ end of the blocked nucleic acidmolecule and one bulky modification located on the 3′ end of the blockednucleic acid molecule, and where the 5′ and 3′ ends comprising the twobulky modifications are less than 11 nm from one another.
 8. The methodof claim 1, wherein blocked nucleic acid molecules include bulkymodifications and wherein the bulky modification is on a 5′ end ofblocked nucleic acid molecules.
 9. The method of claim 1, whereinblocked nucleic acid molecules include bulky modifications and whereinthe bulky modification is on a 3′ end of the blocked nucleic acidmolecules.
 10. The method of claim 1, wherein blocked nucleic acidmolecules include bulky modifications and wherein the bulky modificationis between two internal nucleic acid residues of the blocked nucleicacid molecules.
 11. The method of claim 1, wherein the RNP2s comprise avariant nuclease and the variant nuclease comprises at least onemutation to the PAM-acting domain selected from mutations to amino acidresidues K538, Y542 and K595 in relation to SEQ ID NO:1 and equivalentamino acid residues in orthologs.
 12. The method of claim 11, whereinthere are at least two mutations to the PAM-acting domain selected frommutations to amino acid residues K538, Y542 and K595 in relation to SEQID NO:1 and equivalent amino acid residues in orthologs.
 13. The methodof claim 12, wherein there are at least three mutations to thePAM-acting domain selected from mutations to amino acid residues K538,Y542 and K595 in relation to SEQ ID NO:1 and equivalent amino acidresidues in orthologs.
 14. The method of claim 1, wherein the RNP2scomprise a variant nuclease and the variant nuclease comprises at leastone mutation to the PAM-acting domain of the variant nucleic acid-guidednuclease and wherein the at least one mutation is selected frommutations to amino acid residues K548, N552 and K607 in relation to SEQID NO:2; mutations to amino acid residues K534, Y538 and R591 inrelation to SEQ ID NO:3; mutations to amino acid residues K541, N545 andK601 in relation to SEQ ID NO:4; mutations to amino acid residues K579,N583 and K635 in relation to SEQ ID NO:5; mutations to amino acidresidues K613, N617 and K671 in relation to SEQ ID NO:6; from mutationsto amino acid residues K613, N617 and K671 in relation to SEQ ID NO:7;mutations to amino acid residues K617, N621 and K678 in relation to SEQID NO:8; mutations to amino acid residues K541, N545 and K601 inrelation to SEQ ID NO:9; mutations to amino acid residues K569, N573 andK625 in relation to SEQ ID NO:10; mutations to amino acid residues K562,N566 and K619 in relation to SEQ ID NO:11; mutations to amino acidresidues K645, N649 and K732 in relation to SEQ ID NO:12; mutations toamino acid residues K548, N552 and K607 in relation to SEQ ID NO:13;mutations to amino acid residues K592, N596 and K653 in relation to SEQID NO:14; or mutations to amino acid residues K521, N525 and K577 inrelation to SEQ ID NO:15.
 15. The method of claim 1, wherein the RNP2scomprise a variant nucleic acid-guided nuclease comprising at least onemutation to the domains that interact with the PAM region or surroundingsequences on the blocked nucleic acid molecules and wherein singlestranded DNA is cleaved at least two times faster than double strandedDNA is cleaved.
 16. The method of claim 1, wherein the plurality ofblocked nucleic acid molecules and the RNP2s are at a molarconcentration of at least 2 blocked nucleic acids to 1 RNP2 in thereaction mixture.
 17. The method of claim 1, wherein the target nucleicacid molecule of interest is of bacterial or viral origin.
 18. Themethod of claim 1, wherein the target nucleic acid molecule of interestis from a human or other animal.
 19. The method of claim 18, wherein thesample is selected from blood, plasma, serum, urine, stool, sputum,mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion,seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, atransudate, an exudate, or fluid obtained from a joint, or a swab ofskin or mucosal membrane surface.
 20. The method of claim 21, whereinthe sample is a blood sample from a transplant patient and the targetnucleic acid molecule is a donor-derived genomic sequence.
 21. Themethod of claim 21, wherein the sample is a blood sample from atransplant patient and the target nucleic acid molecules are ahemoglobin S gene and a hemoglobin C gene.
 22. The method of claim 20,wherein the target nucleic acid molecule is a pathogen that infectslivestock.
 23. The method of claim 1, wherein the sample is anenvironmental sample.
 24. The method of claim 23, wherein the sample isselected from the group of a soil sample, an air sample, and a watersample.
 25. The method of claim 24, wherein the sample is a sewersample.
 26. The method of claim 1, wherein the target nucleic acidmolecule is a pathogen used as a bioweapon.
 27. The method of claim 20,wherein the target nucleic acid is a human biomarker.
 28. The method ofclaim 27, wherein the human biomarker is a cancer biomarker.
 29. Themethod of claim 1, wherein there are at least ten target nucleic acidmolecules of interest in the sample.
 30. The method of claim 29, whereinthere are at least twenty target nucleic acid molecules of interest inthe sample.